Artificial self-sufficient cytochrome p450s

ABSTRACT

The disclosure relates to the field of fusion proteins. In some aspects, the invention to artificial fusion proteins comprising cytochrome P450 enzymes linked to reductase enzymes and uses thereof. In some aspects, the disclosures relates to compounds produced by artificial cytochrome P450 enzymes.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S.application Ser. No. 15/552,081, filed Aug. 18, 2017, entitled“ARTIFICIAL SELF-SUFFICIENT CYTOCHROME p450s”, which is a national stagefiling under 35 U.S.C. § 371 of international applicationPCT/US2016/018470, filed Feb. 18, 2016, which was published under PTCArticle 21(2) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/118,445, filed Feb. 19,2015, and U.S. Provisional Patent Application No. 62/275,018, filed Jan.5, 2016, the entire disclosures of each of which are incorporated byreference herein.

BACKGROUND OF INVENTION

Nitro (—NO₂) compounds, particularly nitro aromatic and heterocyclicderivatives, are important industrial chemicals, with an estimatedannual production of greater than 108 tons (Kulkarni and Chaudhari2007). Their applications span a broad range such as food additives,pesticides, herbicides, polymers, explosives, and dyes (Ju and Parales2010). The nitro group is also an important functional unit inpharmaceuticals such as chloramphenicol, nilutamine, tolcapone,metronidazole, and the recently approved anti-tuberculosis drugdelamanid (Martino et al. 2003). Its therapeutic relevance is furtherillustrated by nitro-containing lead drug candidates such as9-nitro-noscapine for the treatment of multidrug resistant cancers(Aneja et al. 2006) and 5-nitro-2-furancarboxylamides in treatingneglected parasitic protozoa infections (Zhou et al. 2013).

Aromatic nitration is a widely used organic reaction (Yan and Yang2013). Industrial scale reactions usually include a mixture of nitricacid and sulfuric acid or sometimes nitric acid with other acids. Inthese reactions, the nitronium ion, NO₂ ⁺, is believed to be the activespecies, albeit the potential minor contribution of a radical mechanism(Olah et al. 1978). Currently used methods and materials present severalchallenges, such as poor selectivity, low yield, generation of multipleisomers and by-products, and low functional group tolerance frequentlyoccur and limit their uses in generating products with specificrequirements. In addition, currently used methods are notenvironmentally sound. Accordingly, there is a need to developenvironmentally benign, selective, practical and efficient directaromatic nitration approaches.

SUMMARY OF INVENTION

Aromatic nitration, addition of a nitro (NO₂) group to an aromaticmolecule, is an important chemical reaction in a variety of industries.Current industrial methods of aromatic nitration utilize chemicalcatalysts, for example the mixing of strong acids (e.g. nitric acid andsulfuric acid). However, this approach is inefficient, leading to lowyield of desirable products, as well as environmentally unsound.

The instant invention, in some aspects, overcomes these issues byproviding a biocatalyst-based approach for aromatic nitration. Thedisclosure is based, in part, on the inventors' unexpected discoverythat a cytochrome P450 enzyme, and in particular artificialself-sufficient cytochrome P450 enzymes, can transfer a nitro group ontoL-tryptophan or L-tryptophan-containing moieties (e.g., a compound ofFormulae Ia-IXa) having a substituted indole ring efficiently and withhigh regio-selectivity. It also was discovered unexpectedly that theregio-selectivity can be altered depending on the particular substitutedL-tryptophan used as a starting material. Thus, the invention providesnovel enzymes, novel methods, novel substituted indoles and novelsubstituted L-tryptophan-containing compounds (e.g., compounds ofFormulae I, Ia, II, IIa, III, IIIa, IV, IVa, V, Va, VI, VIa, VII, VIIa,VIII, VIIIa, IX, or IXa).

Accordingly, in some aspects the disclosure relates to artificialself-sufficient cytochrome P450 enzymes. In some embodiments, artificialself-sufficient cytochrome P450 enzymes are fusion proteins. In someaspects, the disclosure provides a fusion protein comprising (i) acytochrome P450 enzyme which catalyzes transfer of a nitro functionalgroup to a L-tryptophan having at least one substitution on its indolering; (ii) an amino acid linker; and, (iii) a catalytic domain of areductase enzyme; wherein the linker joins the reductase enzyme to aterminus of the cytochrome P450 enzyme.

Accordingly, in some aspects the disclosure relates to artificialself-sufficient cytochrome P450 enzymes. In some embodiments, artificialself-sufficient cytochrome P450 enzymes are fusion proteins. In someaspects, the disclosure provides a fusion protein comprising (i) acytochrome P450 enzyme which catalyzes transfer of a nitro functionalgroup to a compound of Formulae Ia-IXa; (ii) an amino acid linker; and,(iii) a catalytic domain of a reductase enzyme; wherein the linker joinsthe reductase enzyme to a terminus of the cytochrome P450 enzyme.

In some embodiments, the terminus of the cytochrome P450 enzyme is aC-terminus. In some embodiments, the P450 enzyme occurs naturally inStreptomyces. In some embodiments, the P450 enzyme is a TxtE enzyme,wherein a TxtE enzyme is defined as:

-   -   (i) TxtE;    -   (ii) a portion of TxtE which catalyzes transfer of a nitro        functional group to a L-tryptophan having at least one        substitution on its indole ring; or,    -   (iii) an enzyme which catalyzes transfer of a nitro functional        group to a L-tryptophan having at least one substitution on its        indole ring and is at least 95% homologous to the amino acid        sequence of TxtE.

In some embodiments, the terminus of the cytochrome P450 enzyme is aC-terminus. In some embodiments, the P450 enzyme occurs naturally inStreptomyces. In some embodiments, the P450 enzyme is a TxtE enzyme,wherein a TxtE enzyme is defined as:

-   -   (i) TxtE;    -   (ii) a portion of TxtE which catalyzes transfer of a nitro        functional group to a compound of Formulae Ia-IXa; or,    -   (iii) an enzyme which catalyzes transfer of a nitro functional        group to a compound of Formulae Ia-IXa and is at least 95%        homologous to the amino acid sequence of TxtE.

In some embodiments, the cytochrome P450 enzyme shares at least 90%amino acid sequence similarity with TxtE.

In some embodiments, the reductase enzyme is a prokaryotic reductaseenzyme. In some embodiments, the prokaryotic reductase enzyme occursnaturally in a self-sufficient cytochrome P450. In some embodiments, theprokaryotic reductase enzyme occurs naturally in a class II or class IIIcytochrome P450. In some embodiments, the prokaryotic reductase is aCYP102A1 (P450BM3) reductase or a P450RhF reductase.

In some embodiments, the amino acid linker ranges from about 6 aminoacids to about 16 amino acids in length. In some embodiments, the aminoacid linker is selected from the group consisting of flexible amino acidlinker, rigid amino acid linker and cleavable amino acid linker.

In some aspects, the disclosure relates to an expression constructcomprising a nucleic acid encoding a fusion protein as described by thedisclosure. In some aspects, the disclosure provides an isolated nucleicacid encoding a fusion protein as described by the disclosure. In someaspects, the disclosure provides a host cell comprising an expressionconstruct as described by the disclosure or an isolated nucleic acid asdescribed by the disclosure.

In some aspects, the disclosure relates to a method for producing anitro-substituted indole, the method comprising contacting anL-tryptophan molecule having at least one substitution on its indolering, in the presence of NAD(P)H, with (i) at least one reductaseenzyme; and, (ii) a cytochrome P450 enzyme that catalyzes transfer of anitro functional group to the indole of the L-tryptophan having at leastone substitution on its indole ring. In aspects the L-tryptophan havingat least one substitution on its indole ring is substituted with otherthan a nitro group. In aspects the L-tryptophan molecule having at leastone substitution on its indole ring is singly-substituted on its indolering and the resulting nitro-substituted L-tryptophan is adi-substituted nitro indole. In aspects the method further comprisesisolating the nitrated L-tryptophan. In aspects the method furthercomprises isolating the di-substituted nitrated indole portion of theL-tryptophan molecule from the L-tryptophan molecule.

In some aspects, the disclosure relates to a method for producing anitro-substituted indole, the method comprising contacting a compound ofFormulae Ia-IXa, in the presence of NAD(P)H, with (i) at least onereductase enzyme; and, (ii) a cytochrome P450 enzyme that catalyzestransfer of a nitro functional group to the compound of Formulae Ia-IXa.In aspects the compound of Formulae Ia-IXa is substituted with a moietyother than a nitro group. In aspects the compound of Formulae Ia-IXa issingly-substituted on its indole ring and the resulting compound ofFormulae I-IX is a di-substituted nitro tryptophan. In aspects themethod further comprises isolating the compound of Formulae I-IX. Inaspects the method further comprises isolating the di-substitutednitrated indole portion of the compound of Formulae I-IX from thecompound of Formulae I-IX.

In some embodiments, the cytochrome P450 enzyme and the reductase enzymeare linked by an amino acid linker to form a fusion protein prior tocontacting the indole-substituted L-tryptophan molecule. In someembodiments, the amino acid linker links the reductase enzyme to aterminus of the cytochrome P450 enzyme. In some embodiments, theterminus is a C-terminus.

In some embodiments of the method, the P450 enzyme occurs naturally inStreptomyces.

In some embodiments, the cytochrome P450 enzyme is a TxtE enzyme,wherein a TxtE enzyme is defined as:

-   -   (i) TxtE;    -   (ii) a portion of TxtE which catalyzes transfer of a nitro        functional group to a L-tryptophan having at least one        substitution on its indole ring; or,    -   (iii) an enzyme that catalyzes transfer of a nitro functional        group to a L-tryptophan having at least one substitution on its        indole ring and is at least 95% homologous to the amino acid        sequence of TxtE.

In some embodiments of the method, the P450 enzyme occurs naturally inStreptomyces. In some embodiments, the cytochrome P450 enzyme is a TxtEenzyme, wherein a TxtE enzyme is defined as:

-   -   (i) TxtE;    -   (ii) a portion of TxtE which catalyzes transfer of a nitro        functional group to a compound of Formulae Ia-IXa; or,    -   (iii) an enzyme that catalyzes transfer of a nitro functional        group to a compound of Formulae Ia-IXa and is at least 95%        homologous to the amino acid sequence of TxtE.

In some embodiments of the method, the at least one reductase enzyme isferredoxin reductase. In some embodiments of the method, the ferredoxinreductase is spinach ferredoxin reductase.

In some embodiments, the method further comprises contacting thesubstituted L-tryptophan molecule with a ferredoxin protein in thepresence of NAD(P)H. In some embodiments, the ferredoxin protein isspinach ferredoxin protein.

In some embodiments, the method further comprises contacting thecompound of Formulae Ia-IXa with a ferredoxin protein in the presence ofNAD(P)H. In some embodiments, the ferredoxin protein is spinachferredoxin protein.

In some embodiments, the reductase is a prokaryotic reductase enzyme. Insome embodiments, the prokaryotic reductase enzyme occurs naturally in aself-sufficient cytochrome P450. In some embodiments, the prokaryoticreductase enzyme occurs naturally in a class II or class III cytochromeP450. In some embodiments, the prokaryotic reductase is a CYP102A1(P450BM3) reductase or a P450RhF reductase.

In some embodiments, the amino acid linker ranges from about 6 aminoacids to about 16 amino acids in length. In some embodiments, the aminoacid linker is selected from the group consisting of flexible amino acidlinker, rigid amino acid linker and cleavable amino acid linker.

In some aspects, the disclosure provides compounds produced by nitrationof L-tryptophan. In some embodiments, the L-tryptophan has asubstitution on its indole ring. Accordingly, in some aspects thedisclosure relates to a compound represented by Formula I or Formula II.

In some aspects, the disclosure provides compounds produced by nitrationof L-tryptophan. In some aspects, the disclosure provides compoundsproduced by nitration of a compound of Formulae Ia-IXa to afford acompound of Formulae I-IX. In some embodiments, at least one of X¹, X²,or X³ in Formula Ia, IVa, or Va or at least one of Y¹, Y², or Y³ inFormulae IIa, IIIa, VIa, VIIa, VIIIa, or IXa, is not hydrogen.Accordingly, in some aspects the disclosure relates to a compoundrepresented by Formulae I-IX.

In embodiments, the compounds of the invention include

wherein, in Formula I:X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala),

wherein R^(Ala) is independently hydrogen, substituted or unsubstitutedacyl, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring andwherein the Ala of —OR^(Ala) is not H;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein R^(Ala) is independently hydrogen, substituted or unsubstitutedacyl, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring andwherein the Ala of —OR^(Ala) is not H; and

Y is NO₂.

In embodiments, the disclosure is directed to a compound of Formula I,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; and

Y is NO₂.

In embodiments, the disclosure is directed to a compound of Formula IV,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; and

Y is NO₂.

In embodiments, the disclosure is directed to a compound of Formula V,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl, substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

R₁ is H or optionally substituted alkyl;R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl; andY is NO₂. In another aspect, R₁ is H or alkyl. In another aspect, R₁ isH. In another aspect, R₁ is alkyl. In another aspect, R₁ is H methyl. Inanother aspect, R₂ is H. In another aspect, R₁ and R₂ are each H. Inanother aspect, R₁ is alkyl and R₂ is H. In another aspect, R₁ is methyland R₂ is H.

In embodiments of Formula I, one of X¹, X², or X³ is halogen. Inembodiments the halogen is fluorine. In embodiments of Formula I, X¹,X², or X³ is unsubstituted C₁-C₆ alkyl. In embodiments the unsubstitutedC₁-C₆ alkyl is methyl (—CH₃). In embodiments of Formula I, X¹ ishalogen. In embodiments the halogen is fluorine. In embodiments ofFormula I, X¹ is unsubstituted C₁-C₆ alkyl. In embodiments theunsubstituted C₁-C₆ alkyl is methyl (—CH₃). In embodiments, X² and X³are hydrogen.

In some aspects, the compound disclosure relates to a compound ofFormulae I, IV, or V, wherein at least one of X¹, X², or X³ is a “weaklydeactivating group”, a “weakly activating group”, a “moderatelyactivating group”, or a “strongly activating group”, as known in the artand as defined herein. In other aspects, at least one of X¹, X², or X³is H, halogen (e.g. F, Cl, Br, I), substituted or unsubstituted C₁₋₆alkyl (e.g. methyl, CH₃), substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala).

In another aspect, X¹ is halogen (e.g. F, Cl, Br, I), substituted orunsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃), substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala); and X² and X³ are each independently H, halogen (e.g. F, Cl,Br, I), substituted or unsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃),substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala). In another aspect, X¹ is halogen or C₁₋₆alkyl (e.g. methyl, CH₃). In another aspect, X¹ is halogen. In anotheraspect, X¹ is C₁₋₆ alkyl (e.g. methyl, CH₃). In another aspect, X¹ ishalogen or C₁₋₆ alkyl (e.g. methyl, CH₃) and at least one of X² and X³is hydrogen. In another aspect, X¹ is halogen and each of X² and X³ ishydrogen. In another aspect, X¹ if fluorine and each of X² and X³ ishydrogen. In another aspect, X¹ is C₁₋₆ alkyl and each of X² and X³ ishydrogen. In another aspect, X¹ is methyl and each of X² and X³ ishydrogen.

In embodiments, the compounds of the invention include

wherein, in Formula II:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein R^(Ala) is independently hydrogen, substituted or unsubstitutedacyl, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring andwherein the Ala of —OR^(Ala) is not H; and

X is NO₂, provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In embodiments, the disclosure is directed to a compound of Formula II,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; and

X is NO₂, provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In embodiments, the disclosure is directed to a compound of Formula VI,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; and

X is NO₂, provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In embodiments, the disclosure is directed to a compound of Formula VII,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

R₁ is H or optionally substituted alkyl;

R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl; and

X is NO₂, provided that at least one of Y¹, Y², and Y³ is not hydrogen.In another aspect, R₁ is H or alkyl. In another aspect, R₁ is H. Inanother aspect, R₁ is alkyl. In another aspect, R₁ is H methyl. Inanother aspect, R₂ is H. In another aspect, R₁ and R₂ are each H. Inanother aspect, R₁ is alkyl and R₂ is H. In another aspect, R₁ is methyland R₂ is H.

In embodiments, the disclosure is directed to a compound of Formula III,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; and

X is NO₂.

In embodiments, the disclosure is directed to a compound of FormulaVIII, or a pharmaceutically acceptable salt, prodrug, hydrate, orsolvate thereof:

wherein:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring; and

X is NO₂.

In embodiments, the disclosure is directed to a compound of Formula IX,or a pharmaceutically acceptable salt, prodrug, hydrate, or solvatethereof:

wherein:each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

R₁ is H or optionally substituted alkyl;R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl; andX is NO₂. In another aspect, R₁ is H or alkyl. In another aspect, R₁ isH. In another aspect, R₁ is alkyl. In another aspect, R₁ is H methyl. Inanother aspect, R₂ is H. In another aspect, R₁ and R₂ are each H. Inanother aspect, R₁ is alkyl and R₂ is H. In another aspect, R₁ is methyland R₂ is H.

In embodiments, Y¹, Y², or Y³ is halogen and the halogen is fluorine. Inembodiments, Y¹, Y², or Y³ is unsubstituted C₁-C₆ alkyl. In embodiments,the unsubstituted C₁-C₆ alkyl is methyl (—CH₃). In embodiments, two ofY¹, Y² and Y³ are hydrogen. In embodiments, Y² and Y³ are hydrogen. Inembodiments, Y¹ and Y³ are hydrogen. In embodiments, Y¹ and Y² arehydrogen.

In some aspects, the disclosure relates to a compound of Formulae II-IX,wherein at least one of Y¹, Y² or Y³ is halogen or C₁₋₆ alkyl (e.g.methyl, CH₃). In another aspect, Y³ is halogen (e.g. F, Cl, Br, I),substituted or unsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃), substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala); and Y¹ and Y² are each independently H,halogen (e.g. F, Cl, Br, I), substituted or unsubstituted C₁₋₆ alkyl(e.g. methyl, CH₃), substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), N(R^(Ala))₂, or —SR^(Ala). In another aspect, Y³is halogen or C₁₋₆ alkyl (e.g. methyl, CH₃). In another aspect, Y³ ishalogen or C₁₋₆ alkyl (e.g. methyl, CH₃) and at least one of Y¹ and Y²is hydrogen. In another aspect, Y³ is halogen or C₁₋₆ alkyl (e.g.methyl, CH₃) and Y¹ and Y² are each hydrogen. In another aspect, Y³ ishalogen. In another aspect, Y³ is halogen and at least one of Y¹ and Y²is hydrogen. In another aspect, Y³ is halogen and Y¹ and Y² are eachhydrogen. In certain embodiments, Y³ is fluorine and at least one of Y¹and Y² is hydrogen. In another aspect, Y³ is fluorine and Y¹ and Y² areeach hydrogen. In another aspect, Y³ is C₁₋₆ alkyl. In another aspect,Y³ is C₁₋₆ alkyl and at least one of Y¹ and Y² is hydrogen. In anotheraspect, Y³ is C₁₋₆ alkyl and Y¹ and Y² are each hydrogen. In certainembodiments, Y³ is methyl and at least one of Y¹ and Y² is hydrogen. Inanother aspect, Y³ is methyl and Y¹ and Y² are each hydrogen.

In another aspect, the invention is directed to a compound that is:

-   (S)-2-amino-3-(5-methyl-4-nitro-1H-indol-3-yl)propanoic acid (8);-   (S)-2-amino-3-(6-methyl-4-nitro-1H-indol-3-yl)propanoic acid (9);-   (S)-2-amino-3-(7-methyl-4-nitro-1H-indol-3-yl)propanoic acid (10);-   (S)-2-amino-3-(4-methyl-7-nitro-1H-indol-3-yl)propanoic acid (11);-   (S)-2-amino-3-(5-fluoro-4-nitro-1H-indol-3-yl)propanoic acid;-   (S)-2-amino-3-(6-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (12);-   (S)-2-amino-3-(7-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (13);-   (S)-2-amino-3-(4-fluoro-7-nitro-1H-indol-3-yl)propanoic acid;-   (S)-2-amino-3-(5-chloro-4-nitro-1H-indol-3-yl)propanoic acid (14);-   (S)-2-amino-3-(6-chloro-4-nitro-1H-indol-3-yl)propanoic acid (15);-   (S)-2-amino-3-(7-chloro-4-nitro-1H-indol-3-yl)propanoic acid (16);-   (S)-2-amino-3-(4-chloro-7-nitro-1H-indol-3-yl)propanoic acid (17);-   (S)-2-amino-3-(5-bromo-4-nitro-1H-indol-3-yl)propanoic acid (18);-   (S)-2-amino-3-(6-bromo-4-nitro-1H-indol-3-yl)propanoic acid (19);-   (S)-2-amino-3-(7-bromo-4-nitro-1H-indol-3-yl)propanoic acid (20);-   (S)-2-amino-3-(4-bromo-7-nitro-1H-indol-3-yl)propanoic acid (21);-   (S)-2-amino-3-(5-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (22);-   (S)-2-amino-3-(6-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (23);-   (S)-2-amino-3-(7-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (24);-   (S)-2-amino-3-(4-methoxy-7-nitro-1H-indol-3-yl)propanoic acid (25);-   (S)-2-amino-3-(5-amino-4-nitro-1H-indol-3-yl)propanoic acid (26);-   (S)-2-amino-3-(6-amino-4-nitro-1H-indol-3-yl)propanoic acid (27);-   (S)-2-amino-3-(7-amino-4-nitro-1H-indol-3-yl)propanoic acid (28);-   (S)-2-amino-3-(4-amino-7-nitro-1H-indol-3-yl)propanoic acid (29);-   (S)-2-amino-3-(5-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (30);-   (S)-2-amino-3-(6-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (31);-   (S)-2-amino-3-(7-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (32);-   (S)-2-amino-3-(4-hydroxy-7-nitro-1H-indol-3-yl)propanoic acid (33);-   (S)-2-amino-3-(4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid (34);-   (S)-2-amino-3-(4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid (35);-   (S)-2-amino-3-(4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid (36);-   (S)-2-amino-3-(7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid (37);-   (S)-2-amino-3-(5-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid    (38);-   (S)-2-amino-3-(6-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid    (39);-   (S)-2-amino-3-(7-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid    (40);-   (S)-2-amino-3-(4-cyclopropyl-7-nitro-1H-indol-3-yl)propanoic acid    (41);-   (S)-2-amino-3-(4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid (42);-   (S)-2-amino-3-(4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid (43);-   (S)-2-amino-3-(4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid (44);-   (S)-2-amino-3-(7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid (45);-   (S)-2-amino-3-(5-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (46);-   (S)-2-amino-3-(6-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (47);-   (S)-2-amino-3-(7-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (48);-   (S)-2-amino-3-(4-ethynyl-7-nitro-1H-indol-3-yl)propanoic acid (49);-   (S)-2-amino-3-(5-morpholino-4-nitro-1H-indol-3-yl)propanoic acid    (50);-   (S)-2-amino-3-(6-morpholino-4-nitro-1H-indol-3-yl)propanoic acid    (51);-   (S)-2-amino-3-(7-morpholino-4-nitro-1H-indol-3-yl)propanoic acid    (52);-   (S)-2-amino-3-(4-morpholino-7-nitro-1H-indol-3-yl)propanoic acid    (53);-   (S)-2-amino-3-(5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid    (54);-   (S)-2-amino-3-(6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid    (55);-   (S)-2-amino-3-(7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid    (56);-   (S)-2-amino-3-(4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic acid    (57);-   (S)-2-amino-3-(4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (58);-   (S)-2-amino-3-(4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (59);-   (S)-2-amino-3-(4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (60);-   (S)-2-amino-3-(7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (61);-   2-amino-3-(5-methyl-4-nitro-1H-indol-3-yl)propanoic acid (62);-   2-amino-3-(6-methyl-4-nitro-1H-indol-3-yl)propanoic acid (63);-   2-amino-3-(7-methyl-4-nitro-1H-indol-3-yl)propanoic acid (64);-   2-amino-3-(4-methyl-7-nitro-1H-indol-3-yl)propanoic acid (65);-   2-amino-3-(6-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (66);-   2-amino-3-(7-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (67);-   2-amino-3-(4-fluoro-7-nitro-1H-indol-3-yl)propanoic acid (68);-   2-amino-3-(5-chloro-4-nitro-1H-indol-3-yl)propanoic acid (69);-   2-amino-3-(6-chloro-4-nitro-1H-indol-3-yl)propanoic acid (70);-   2-amino-3-(7-chloro-4-nitro-1H-indol-3-yl)propanoic acid (71);-   2-amino-3-(4-chloro-7-nitro-1H-indol-3-yl)propanoic acid (72);-   2-amino-3-(5-bromo-4-nitro-1H-indol-3-yl)propanoic acid (73);-   2-amino-3-(6-bromo-4-nitro-1H-indol-3-yl)propanoic acid (74);-   2-amino-3-(7-bromo-4-nitro-1H-indol-3-yl)propanoic acid (75);-   2-amino-3-(4-bromo-7-nitro-1H-indol-3-yl)propanoic acid (76);-   2-amino-3-(5-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (77);-   2-amino-3-(6-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (78);-   2-amino-3-(7-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (79);-   2-amino-3-(4-methoxy-7-nitro-1H-indol-3-yl)propanoic acid (80);-   2-amino-3-(5-amino-4-nitro-1H-indol-3-yl)propanoic acid (81);-   2-amino-3-(6-amino-4-nitro-1H-indol-3-yl)propanoic acid (82);-   2-amino-3-(7-amino-4-nitro-1H-indol-3-yl)propanoic acid (83);-   2-amino-3-(4-amino-7-nitro-1H-indol-3-yl)propanoic acid (84);-   2-amino-3-(5-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (85);-   2-amino-3-(6-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (86);-   2-amino-3-(7-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (87);-   2-amino-3-(4-hydroxy-7-nitro-1H-indol-3-yl)propanoic acid (88);-   2-amino-3-(4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid (89);-   2-amino-3-(4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid (90);-   2-amino-3-(4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid (91);-   2-amino-3-(7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid (92);-   2-amino-3-(5-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (93);-   2-amino-3-(6-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (94);-   2-amino-3-(7-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (95);-   2-amino-3-(4-cyclopropyl-7-nitro-1H-indol-3-yl)propanoic acid (96);-   2-amino-3-(4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid (97);-   2-amino-3-(4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid (98);-   2-amino-3-(4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid (99);-   2-amino-3-(7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid (100);-   2-amino-3-(5-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (101);-   2-amino-3-(6-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (102);-   2-amino-3-(7-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (103);-   2-amino-3-(4-ethynyl-7-nitro-1H-indol-3-yl)propanoic acid (104);-   2-amino-3-(5-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (105);-   2-amino-3-(6-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (106);-   2-amino-3-(7-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (107);-   2-amino-3-(4-morpholino-7-nitro-1H-indol-3-yl)propanoic acid (108);-   2-amino-3-(5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid    (109);-   2-amino-3-(6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid    (110);-   2-amino-3-(7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid    (111);-   2-amino-3-(4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic acid    (112);-   2-amino-3-(4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (113);-   2-amino-3-(4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (114);-   2-amino-3-(4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (115);-   2-amino-3-(7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid    (116);-   2-amino-3-(1,5-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (117);-   2-amino-3-(1,6-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (118);-   2-amino-3-(1,7-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (119);-   2-amino-3-(1,4-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid (120);-   2-amino-3-(6-fluoro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (121);-   2-amino-3-(7-fluoro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (122);-   2-amino-3-(4-fluoro-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (123);-   2-amino-3-(5-chloro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (124);-   2-amino-3-(6-chloro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (125);-   2-amino-3-(7-chloro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (126);-   2-amino-3-(4-chloro-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (127);-   2-amino-3-(5-bromo-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (128);-   2-amino-3-(6-bromo-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (129);-   2-amino-3-(7-bromo-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (130);-   2-amino-3-(4-bromo-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (131);-   2-amino-3-(5-methoxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (132);-   2-amino-3-(6-methoxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (133);-   2-amino-3-(7-methoxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (134);-   2-amino-3-(4-methoxy-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (135);-   2-amino-3-(5-amino-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (136);-   2-amino-3-(6-amino-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (137);-   2-amino-3-(7-amino-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (138);-   2-amino-3-(4-amino-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (139);-   2-amino-3-(5-hydroxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (140);-   2-amino-3-(6-hydroxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (141);-   2-amino-3-(7-hydroxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (142);-   2-amino-3-(4-hydroxy-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (143);-   2-amino-3-(1-methyl-4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid    (144);-   2-amino-3-(1-methyl-4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid    (145);-   2-amino-3-(1-methyl-4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid    (146);-   2-amino-3-(1-methyl-7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid    (147);-   2-amino-3-(5-cyclopropyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic    acid (148);-   2-amino-3-(6-cyclopropyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic    acid (149);-   2-amino-3-(7-cyclopropyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic    acid (150);-   2-amino-3-(4-cyclopropyl-1-methyl-7-nitro-1H-indol-3-yl)propanoic    acid (151);-   2-amino-3-(1-methyl-4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid    (152);-   2-amino-3-(1-methyl-4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid    (153);-   2-amino-3-(1-methyl-4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid    (154);-   2-amino-3-(1-methyl-7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid    (155);-   2-amino-3-(5-ethynyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (156);-   2-amino-3-(6-ethynyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (157);-   2-amino-3-(7-ethynyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (158);-   2-amino-3-(4-ethynyl-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (159);-   2-amino-3-(1-methyl-5-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (160);-   2-amino-3-(1-methyl-6-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (161);-   2-amino-3-(1-methyl-7-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (162);-   2-amino-3-(1-methyl-4-morpholino-7-nitro-1H-indol-3-yl)propanoic    acid (163);-   2-amino-3-(1-methyl-5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (164);-   2-amino-3-(1-methyl-6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (165);-   2-amino-3-(1-methyl-7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (166);-   2-amino-3-(1-methyl-4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic    acid (167);-   2-amino-3-(1-methyl-4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (168);-   2-amino-3-(1-methyl-4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (169);-   2-amino-3-(1-methyl-4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (170);-   2-amino-3-(1-methyl-7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (171);-   2-amino-3-(2,5-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (172);-   2-amino-3-(2,6-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (173);-   2-amino-3-(2,7-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (174);-   2-amino-3-(2,4-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid (175);-   2-amino-3-(6-fluoro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (176);-   2-amino-3-(7-fluoro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (177);-   2-amino-3-(4-fluoro-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (178);-   2-amino-3-(5-chloro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (179);-   2-amino-3-(6-chloro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (180);-   2-amino-3-(7-chloro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (181);-   2-amino-3-(4-chloro-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (182);-   2-amino-3-(5-bromo-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (183);-   2-amino-3-(6-bromo-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (184);-   2-amino-3-(7-bromo-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (185);-   2-amino-3-(4-bromo-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (186);-   2-amino-3-(5-methoxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (187);-   2-amino-3-(6-methoxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (188);-   2-amino-3-(7-methoxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (189);-   2-amino-3-(4-methoxy-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (190);-   2-amino-3-(5-amino-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (191);-   2-amino-3-(6-amino-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (192);-   2-amino-3-(7-amino-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (193);-   2-amino-3-(4-amino-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (194);-   2-amino-3-(5-hydroxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (195);-   2-amino-3-(6-hydroxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (196);-   2-amino-3-(7-hydroxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (197);-   2-amino-3-(4-hydroxy-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (198);-   2-amino-3-(2-methyl-4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid    (199);-   2-amino-3-(2-methyl-4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid    (200);-   2-amino-3-(2-methyl-4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid    (201);-   2-amino-3-(2-methyl-7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid    (202);-   2-amino-3-(5-cyclopropyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic    acid (203);-   2-amino-3-(6-cyclopropyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic    acid (204);-   2-amino-3-(7-cyclopropyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic    acid (205);-   2-amino-3-(4-cyclopropyl-2-methyl-7-nitro-1H-indol-3-yl)propanoic    acid (206);-   2-amino-3-(2-methyl-4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid    (207);-   2-amino-3-(2-methyl-4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid    (208);-   2-amino-3-(2-methyl-4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid    (209);-   2-amino-3-(2-methyl-7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid    (210);-   2-amino-3-(5-ethynyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (211);-   2-amino-3-(6-ethynyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (212);-   2-amino-3-(7-ethynyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid    (213);-   2-amino-3-(4-ethynyl-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid    (214);-   2-amino-3-(2-methyl-5-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (215);-   2-amino-3-(2-methyl-6-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (216);-   2-amino-3-(2-methyl-7-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (217);-   2-amino-3-(2-methyl-4-morpholino-7-nitro-1H-indol-3-yl)propanoic    acid (218);-   2-amino-3-(2-methyl-5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (219);-   2-amino-3-(2-methyl-6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (220);-   2-amino-3-(2-methyl-7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (221);-   2-amino-3-(2-methyl-4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic    acid (222);-   2-amino-3-(2-methyl-4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (223);-   2-amino-3-(2-methyl-4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (224);-   2-amino-3-(2-methyl-4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (225);-   2-amino-3-(2-methyl-7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (226);-   2-amino-3-(1,2,5-trimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (227);-   2-amino-3-(1,2,6-trimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (228);-   2-amino-3-(1,2,7-trimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (229);-   2-amino-3-(1,2,4-trimethyl-7-nitro-1H-indol-3-yl)propanoic acid    (230);-   2-amino-3-(6-fluoro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (231);-   2-amino-3-(7-fluoro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (232);-   2-amino-3-(4-fluoro-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic    acid (233);-   2-amino-3-(5-chloro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (234);-   2-amino-3-(6-chloro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (235);-   2-amino-3-(7-chloro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (236);-   2-amino-3-(4-chloro-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic    acid (237);-   2-amino-3-(5-bromo-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (238);-   2-amino-3-(6-bromo-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (239);-   2-amino-3-(7-bromo-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (240);-   2-amino-3-(4-bromo-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid    (241);-   2-amino-3-(5-methoxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (242);-   2-amino-3-(6-methoxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (243);-   2-amino-3-(7-methoxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (244);-   2-amino-3-(4-methoxy-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic    acid (245);-   2-amino-3-(5-amino-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (246);-   2-amino-3-(6-amino-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (247);-   2-amino-3-(7-amino-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid    (248);-   2-amino-3-(4-amino-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid    (249);-   2-amino-3-(5-hydroxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (250);-   2-amino-3-(6-hydroxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (251);-   2-amino-3-(7-hydroxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (252);-   2-amino-3-(4-hydroxy-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic    acid (253);-   2-amino-3-(1,2-dimethyl-4-nitro-5-phenyl-1H-indol-3-yl)propanoic    acid (254);-   2-amino-3-(1,2-dimethyl-4-nitro-6-phenyl-1H-indol-3-yl)propanoic    acid (255);-   2-amino-3-(1,2-dimethyl-4-nitro-7-phenyl-1H-indol-3-yl)propanoic    acid (256);-   2-amino-3-(1,2-dimethyl-7-nitro-4-phenyl-1H-indol-3-yl)propanoic    acid (257);-   2-amino-3-(5-cyclopropyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (258);-   2-amino-3-(6-cyclopropyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (259);-   2-amino-3-(7-cyclopropyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (260);-   2-amino-3-(4-cyclopropyl-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic    acid (261);-   2-amino-3-(1,2-dimethyl-4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid    (262);-   2-amino-3-(1,2-dimethyl-4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid    (263);-   2-amino-3-(1,2-dimethyl-4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid    (264);-   2-amino-3-(1,2-dimethyl-7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid    (265);-   2-amino-3-(5-ethynyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (266);-   2-amino-3-(6-ethynyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (267);-   2-amino-3-(7-ethynyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic    acid (268);-   2-amino-3-(4-ethynyl-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic    acid (269);-   2-amino-3-(1,2-dimethyl-5-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (270);-   2-amino-3-(1,2-dimethyl-6-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (271);-   2-amino-3-(1,2-dimethyl-7-morpholino-4-nitro-1H-indol-3-yl)propanoic    acid (272);-   2-amino-3-(1,2-dimethyl-4-morpholino-7-nitro-1H-indol-3-yl)propanoic    acid (273);-   2-amino-3-(1,2-dimethyl-5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (274);-   2-amino-3-(1,2-dimethyl-6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (275);-   2-amino-3-(1,2-dimethyl-7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic    acid (276);-   2-amino-3-(1,2-dimethyl-4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic    acid (277);-   2-amino-3-(1,2-dimethyl-4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (278);-   2-amino-3-(1,2-dimethyl-4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (279);-   2-amino-3-(1,2-dimethyl-4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (280); or-   2-amino-3-(1,2-dimethyl-7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic    acid (281);-   and a pharmaceutically acceptable salt, prodrug, hydrate, or solvate    thereof.

In some aspects, the disclosure relates to a composition comprising thecompound of Formula I or Formula II. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier. Insome aspects, the disclosure provides a polypeptide comprising thecompound of Formula I or Formula II. In some aspects, the disclosurerelates to a cell comprising a compound of Formula I or Formula II.

In some aspects, the disclosure relates to methods of producing acompound of Formula I or Formula II. In some aspects, the methodcomprises contacting a L-tryptophan having at least one substitution onits indole ring with (i) at least one reductase enzyme; and, (ii) acytochrome P450 enzyme which catalyzes transfer of a nitro functionalgroup to a L-tryptophan having at least one substitution on its indolering, in the presence of NAD(P)H.

In some aspects, the disclosure relates to a composition comprising thecompound of Formulae I-IX, or a pharmaceutically acceptable salt,prodrug, hydrate, or solvate thereof. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier. Insome aspects, the disclosure provides a polypeptide comprising thecompound of Formulae I-IX. In some aspects, the disclosure relates to acell comprising a compound of Formulae I-IX.

In some aspects, the disclosure relates to methods of producing acompound of Formulae I-IX, or a pharmaceutically acceptable salt,prodrug, hydrate, or solvate thereof. In some aspects, the methodcomprises contacting a compound of Formulae Ia-IXa with (i) at least onereductase enzyme; and, (ii) a cytochrome P450 enzyme which catalyzestransfer of a nitro functional group to a compound of Formulae Ia-IXa,in the presence of NAD(P)H.

In another aspect, the disclosure related to a method of producing acompound of Formula I, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula Ia:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula Ia, in the        presence of NAD(P)H;

to produce a compound of Formula I:

wherein:

each X¹ is independently halogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₂₋₆ alkenyl, substituted orunsubstituted C₂₋₆ alkynyl, substituted or unsubstituted, monocyclic,3-to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic,3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl,substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl,—OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula IV, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IVa:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula Ia, in the        presence of NAD(P)H;

to produce a compound of Formula IV:

wherein:

each X¹ is independently halogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₂₋₆ alkenyl, substituted orunsubstituted C₂₋₆ alkynyl, substituted or unsubstituted, monocyclic,3-to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic,3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl,substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl,—OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula V, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula Va:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula Ia, in the        presence of NAD(P)H;

to produce a compound of Formula V:

wherein:

each X¹ is independently halogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₂₋₆ alkenyl, substituted orunsubstituted C₂₋₆ alkynyl, substituted or unsubstituted, monocyclic,3-to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic,3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl,substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl,—OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

Y is NO₂;

R₁ is H or optionally substituted alkyl;R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl. In another aspect, R₁ is H or alkyl.In another aspect, R₁ is H. In another aspect, R₁ is alkyl. In anotheraspect, R₁ is H methyl. In another aspect, R₂ is H. In another aspect,R₁ and R₂ are each H. In another aspect, R₁ is alkyl and R₂ is H. Inanother aspect, R₁ is methyl and R₂ is H.

In another aspect, the disclosure related to a method of producing acompound of Formula II, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IIa:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula II:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In another aspect, the disclosure related to a method of producing acompound of Formula VI, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula VIa:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula VI:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In another aspect, the disclosure related to a method of producing acompound of Formula VII, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula VIIa:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula VII:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

R₁ is H or optionally substituted alkyl;

R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl;

provided that at least one of Y¹, Y², and Y³ is not hydrogen. In anotheraspect, R₁ is H or alkyl. In another aspect, R₁ is H. In another aspect,R₁ is alkyl. In another aspect, R₁ is H methyl. In another aspect, R₂ isH. In another aspect, R₁ and R₂ are each H. In another aspect, R₁ isalkyl and R₂ is H. In another aspect, R₁ is methyl and R₂ is H.

In another aspect, the disclosure related to a method of producing acompound of Formula III, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof. the method comprising contacting a compoundof Formula IIIa:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIIa, in the        presence of NAD(P)H;

to produce a compound of Formula III:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula VIII, or a pharmaceutically acceptable salt,prodrug, hydrate, or solvate thereof, the method comprising contacting acompound of Formula VIIIa:

with:

-   -   (iii) at least one reductase enzyme; and    -   (iv) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula VIII:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula IX, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IXa:

with:

-   -   (iii) at least one reductase enzyme; and    -   (iv) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula IX:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

X is NO₂;

R₁ is H or optionally substituted alkyl;

R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl. In another aspect, R₁ is H or alkyl.In another aspect, R₁ is H. In another aspect, R₁ is alkyl. In anotheraspect, R₁ is H methyl. In another aspect, R₂ is H. In another aspect,R₁ and R₂ are each H. In another aspect, R₁ is alkyl and R₂ is H. Inanother aspect, R₁ is methyl and R₂ is H.

In some embodiments, the reductase enzyme and the cytochrome P450 enzymeare linked by an amino acid linker to form a fusion protein prior tocontacting the indole-substituted L-tryptophan molecule. In someembodiments, the amino acid linker links reductase enzyme to a terminusof cytochrome P450. In some embodiments, the terminus is a C-terminus.

In some embodiments, the P450 enzyme occurs naturally in Streptomyces.In some embodiments, the cytochrome P450 enzyme is a TxtE enzyme,wherein a TxtE enzyme is defined as:

(i) TxtE;

(ii) a portion of TxtE which catalyzes transfer of a nitro functionalgroup to a L-tryptophan having at least one substitution on its indolering; or,(iii) an enzyme that catalyzes transfer of a nitro functional group to aL-tryptophan having at least one substitution on its indole ring and isat least 95% homologous to the amino acid sequence of TxtE.

In some embodiments, the at least one reductase enzyme is ferredoxinreductase. In some embodiments, the ferredoxin reductase is spinachferredoxin reductase. In some embodiments, the method further comprisescontacting the substituted L-tryptophan molecule with a ferredoxinprotein in the presence of NAD(P)H. In some embodiments, the ferredoxinprotein is spinach ferredoxin protein.

In some embodiments, the P450 enzyme occurs naturally in Streptomyces.In some embodiments, the cytochrome P450 enzyme is a TxtE enzyme,wherein a TxtE enzyme is defined as:

(i) TxtE;

(ii) a portion of TxtE which catalyzes transfer of a nitro functionalgroup to a compound of Formulae Ia-IXa; or,(iii) an enzyme that catalyzes transfer of a nitro functional group to acompound of Formulae Ia-IXa and is at least 95% homologous to the aminoacid sequence of TxtE.

In some embodiments, the at least one reductase enzyme is ferredoxinreductase. In some embodiments, the ferredoxin reductase is spinachferredoxin reductase. In some embodiments, the method further comprisescontacting the compound of Formulae I, Ia, II, IIa, III, IIIa, IV, IVa,V, Va, VI, VIa, VII, VIIa, VIII, VIIIa, IX, or IXa with a ferredoxinprotein in the presence of NAD(P)H. In some embodiments, the ferredoxinprotein is spinach ferredoxin protein.

In some embodiments, the reductase is a prokaryotic reductase enzyme. Insome embodiments, the prokaryotic reductase enzyme occurs naturally in aself-sufficient cytochrome P450. In some embodiments, the prokaryoticreductase enzyme occurs naturally in a class II or class III cytochromeP450. In some embodiments, the prokaryotic reductase is a CYP102A1(P450BM3) reductase or a P450RhF reductase.

In some embodiments, the amino acid linker ranges from about 6 aminoacids to about 16 amino acids in length. In some embodiments, the aminoacid linker is a flexible amino acid linker, a rigid amino acid linker,and/or a cleavable amino acid linker.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows TxtE catalyzes an aromatic nitration reaction on the C4 ofL-tryptophan indole ring. 02 and NO act as co-substrates. Nicotinamideadenine dinucleotide phosphate (NADPH) is consumed and recycled withglucose dehydrogenase (GDH) in the reaction. Wild-type TxtE requiresspinach ferredoxin (Fer) and ferredoxin reductase (Frd) in this reactionwhile created artificial TxtE variants (TxtE fusions) areself-sufficient.

FIGS. 2A-2C show characterization of recombinant TxtE variants. FIG. 2Adepicts SDS-PAGE analysis of TxtE and its self-sufficient variants.Recombinant proteins were purified with a single Ni-NTA affinity column,and showed expected molecular weights (MW). Lane M: protein marker; lane1, TxtE [calculated MW (cal. MW): 46.3 kDa]; lane 2, TxtE-BM3R (cal. MW:112.1 kDa); lane 3: TxtE-RhFRed (cal. MW: 81.8 kDa); lane 4:TxtE-RhFRed* (cal. MW: 82.7 kDa). FIG. 2B shows spectroscopic analysisof TxtE, TxtEBM3R, TxtE-RhFRed, and TxtE-RhFRed*. FIG. 2C shows relativecatalytic activities of recombinant TxtE self-sufficient variants innitrating L-tryptophan. TxtE was used as the control and its activitywas set as 100%. TxtE-BM3R activity was slightly higher than thecontrol, while both TxtE-RhFRed and TxtE-RhFRed* only retained less than15% of TxtE activity.

FIGS. 3A-3B show thermostability and pH dependence of TxtE andTxtE-BM3R. FIG. 3A shows the thermostability of TxtE and TxtE-BM3R. Bothenzymes showed the similar T₅₀ at around 45° C. FIG. 3B shows pHdependence of TxtE and TxtE-BM3R. Both enzymes exhibited the highestactivity at a range of between pH 8.0 and 9.0.

FIGS. 4A-4C depict MS2 spectra. FIG. 4A shows MS2 spectrum of4-nitro-L-tryptophan. FIG. 4B shows MS2 spectrum of nitrated5-F-L-tryptophan. FIG. 4C shows MS2 spectrum and nitrated4-F-DL-tryptophan. The fragmentation pattern in (FIG. 4A) and (FIG. 4B)was the same but it was different between (FIG. 4B) and (FIG. 4C).Compared with those in (FIG. 4A), the C5-F substitution increased them/z values of most ions in (FIG. 4B) by 18 Da. Putative chemicalstructures of ions in grey labeled peaks were shown in FIG. 7.

FIG. 5 shows the effect of pH stability on activity of TxtE andTxtE-BM3R. Both enzymes were incubated in the buffers with pH from 4.5to 9.5 for 15 min and then used in the reactions with 0.5 mML-tryptophan, 1 mM NADP⁺, 1 mM glucose, ˜10 units/mL self-preparedglucose dehydrogenase crude extract, 1 mM NOC-5 in 100 μL of Tris-HClbuffer (100 mM, pH 8.0). For TxtE reactions, 0.43 μM spinach ferredoxinand 0.33 μM spinach ferredoxin-NADP⁺ reductase were included. Thereactions were incubated at 20° C., 300 rpm on a thermostat (Eppendorf)for 30 minutes. All experiments were performed in duplicate.

FIG. 6 shows substrate binding to TxtE and TxtE-BM3R. Fusion of BM3R toTxtE slightly tightened the binding of 4-F-DL-tryptophan and5-F-L-tryptophan to the enzyme. All experiments were performed at leastin duplicate.

FIG. 7 shows putative fragmentation pathways of 4-nitro-L-tryptophan,4-nitro-5-F-L-tryptophan, and 7-nitro-4-F-L-tryptophan. Exact masses ofall putative ions were shown.

FIGS. 8A-8B show high resolution mass spectrometry (HRMS) spectra ofnitrated products in TxtE-BM3R reactions.

FIGS. 9A-9B show ¹H nuclear magnetic resonance (NMR) spectra of nitratedF-tryptophan products.

FIGS. 10A-10B show ¹³C NMR spectra of nitrated F-tryptophan products.

FIGS. 11A-11B show Heteronuclear Single Quantum Coherence (HSQC) NMRspectra of nitrated F-tryptophan products.

FIGS. 12A-12B show HMBC NMR spectra of nitrated F-tryptophan products.

FIGS. 13A-13C show TxtE nitrates the indole C4 of 1-tryptophan (FIG.13A) and 5-F-1-tryptophan (FIG. 13B) and the C7 of 4-F-l-tryptophanindole ring (FIG. 13C). O₂ and NO act as co-substrates. NADPH isconsumed in the reaction and can be recycled with glucose dehydrogenase(GDH). Wild-type TxtE requires spinach Fer and Frd in this reactionwhile created artificial TxtE variants are self-sufficient.

FIGS. 14A-14C show characterization of recombinant TxtE variants. FIG.14A shows SDS-PAGE analysis of TxtE and its self-sufficient variants.Recombinant proteins were purified with a single Ni-NTA affinity column.Lane M: protein marker; lane 1, TxtE [calculated molecular weight (cal.MW): 46.3 kD]; lane 2, TxtE-BM3R (cal. MW: 112.1 kD); lane 3:TxtE-RhFRed (cal. MW: 81.8 kD); lane 4: TxtE-RhFRed* (cal. MW: 82.7 kD).FIG. 14B shows spectroscopic analysis of TxtE, TxtEBM3R, TxtE-RhFRed,and TxtE-RhFRed*. Black lines: P450 absorbance spectra; green dashedlines: CO-oxidized spectra; blue dotted-dashed line: CO-reduced spectra;red dotted lines: CO-reduced difference spectra. FIG. 14C showscatalytic activities of recombinant TxtE self-sufficient variants innitrating 1-tryptophan. TxtE was used as the control.

FIG. 15 shows HPLC analysis of enzyme nitration reaction mixtures with1-Trp as the substrate. The reactions were performed for 2 hours. L-Trpwas eluted at 1.51 min while the product has a retention time of 1.93min.

FIGS. 16A-16B show substrate binding assays. FIG. 16A shows the changesof spin state of heme iron in TxtE, TxtE-BM3R, TxtE-RhFRed andTxtE-RhFRed* induced by different concentrations of substrates. Black:spectra in the absence of substrate; red: spectra induced by 100 μMsubstrates; orange: spectra induced by 200 μM substrate; and blue:spectra induced by 500 μM substrate. All four enzymes responded to thesubstrate binding in a highly similar manner. 1-Tryptophan induced thehighest percentage of heme iron's in the high-spin state, while4-F-dl-tryptophan had the lowest. FIG. 16B shows spectra changes inducedby substrate binding to TxtE, TxtE-BM3R, TxtE-RhFRed and TxtE-RhFRed*.

FIGS. 17A-17B show data related to thermostability (FIG. 17A) and pHdependence (FIG. 17B) of TxtE and TxtE-BM3R. To test enzymethermostability, enzymes were incubated at a series of temperatures (4to 65° C.) for 15 min. After cooling on ice, enzyme solutions werecentrifuged and used in the 1-tryptophan nitration reaction at 20° C.,300 rpm for 30 min. To test pH dependence, 1-tryptophan nitrationreactions were performed in 100 mM Tris-Cl or sodium phosphate bufferswith various pH values (4.5 to 9.5) at 20° C., 300 rpm for 30 min.Products were quantitated by HPLC. All experiments were performed atleast three times.

FIG. 18 shows the pH stability of TxtE and TxtE-BM3R. Both enzymes wereincubated in the buffers with pH from 4.5 to 9.5 for 15 min and thenused in the reactions with 0.5 mM 1-tryptophan, 1 mM NADP⁺, 1 mMglucose, ˜10 units/mL self-prepared glucose dehydrogenase crude extract,1 mM NOC-5 in 100 μL of Tris-HCl buffer (100 mM, pH 8.0). For TxtEreactions, 0.43 μM spinach ferredoxin and 0.33 μM spinachferredoxin-NADP⁺ reductase were included. The reactions were incubatedat 20° C., 300 rpm on a thermostat (Eppendorf) for 30 minutes. Allexperiments were performed in duplicate.

FIG. 19 shows substrate binding to TxtE and TxtE-BM3R. Fusion of BM3R toTxtE slightly tightened the binding of 4-F-dl-tryptophan and5-F-l-tryptophan to the enzyme. All experiments were performed at leastin duplicate.

FIGS. 20A-20C show LC-MS analysis of Marfey's derivatized 1-Trp and4-nitro-l-Trp (FIG. 20A), 5F-1-Trp and 4-nitro-5-F-1-Trp (FIG. 20B), and4F-dl-Trp and nitrated product (FIG. 20C). Blue: ion extract spectra ofMarfey's derivatized tryptophan analogs; Red: ion extract spectra ofMarfey's derivatized nitration product directly from enzyme reactionmixtures; Green: ion extract spectra of Marfey's derivatized, purifiednitration products.

FIGS. 21A-21C show the MS2 spectra of 4-nitro-l-tryptophan (FIG. 21A),nitrated 5-F-1-tryptophan (FIG. 21B), and nitrated 4-F-dl-tryptophan(FIG. 21C). The reaction mixtures were quenched with twice volumes ofmethanol. After centrifugation, 10 al of each sample was used for theLC/MS/MS analysis. Putative chemical structures of ions in red labeledpeaks were shown in FIG. 23.

FIG. 22 shows UV spectra of all three substrates and their correspondingnitrated products as determined by Shimadzu PDA detector coupled withUHPLC system. All compounds have the same maximal absorbance wavelengthat 211 nm.

FIG. 23 shows Putative fragmentation pathways of 4-nitro-l-tryptophan,4-nitro-5-F-1-tryptophan, and 7-nitro-4-F-l-tryptophan. Exact masses ofall putative ions were shown.

FIGS. 24A-24B show HRMS spectra of nitrated products in TxtE-BM3Rreactions.

FIGS. 25A-25B show ¹H NMR spectra of nitrated F-tryptophan products.

FIGS. 26A-26B show ¹³C NMR spectra of nitrated F-tryptophan products.

FIGS. 27A-27B show HSQC NMR spectra of nitrated F-tryptophan products.

FIGS. 28A-28B show HMBC NMR spectra of nitrated F-tryptophan products.

FIGS. 29A-29B show: binding affinities (FIG. 29A); and relativenitration conversions (FIG. 29B) for L-tryptophan and substitutedtryptophan analogs

DETAILED DESCRIPTION OF INVENTION

Aromatic nitration is an essential chemical reaction for the productionof a variety of important industrial chemicals. For example, nitrocompounds are used in the production of food additives, herbicides andpharmaceuticals. However, currently used technologies to performaromatic nitration on an industrial scale are hampered by challengesranging from lack of reaction efficiency to the production ofenvironmentally unfriendly by-products. Therefore, new approaches fordirect aromatic nitration must be developed.

Without wishing to be bound by any particular theory, aromatic nitrationusing biocatalysts offers a number of distinct advantages, such as highefficiency, high degree of selectivity, mild reaction conditions, andenvironmental friendliness, over currently used chemical catalysis.Accordingly, provided herein are methods and compositions for nitrationof aromatic compounds. In some aspects, the present invention relates tothe use of a biocatalyst for aromatic nitration. In some embodiments,the biocatalyst is a cytochrome P450 enzyme. As discussed above, it isbelieved that the active nitration species in the nitration processesdelineated herein is the nitronium ion, NO₂ ⁺. Thus, it is believed thatthe nitration processes presented herein proceed via an electrophilicaromatic substitution mechanism. Therefore, as is well established inthe art for processes involving electrophilic aromatic substitution,substituents on the aromatic system (e.g., X¹, X², X³ in Formulae I, Ia,IV, IVa, V, or Va; and Y¹, Y², Y³ in Formulae II, IIa, III, IIIa, VI,VIa, VII, VIIa, VIII, VIIIa, IX, or IXa) that increase the electrondensity of the aromatic system are well-known in the art as “activatinggroups”, and increase the rate of electrophilic aromatic substitution(e.g., nitration) relative to the unsubstituted aromatic system, whilesubstituents that decrease the electron density of the aromatic systemare well-known in the art as “deactivating groups”, and decrease therate of electrophilic aromatic substitution relative to theunsubstituted aromatic system. The “activating groups” are furtherclassified as “weakly activating groups” (i.e., groups that weaklyincrease reaction rate), “moderately activating groups” (i.e., groupsthat moderately increase reaction rate), and “strongly activatinggroups” (i.e., groups that strongly increase reaction rate), while“deactivating groups” are further classified as “weakly deactivatinggroups” (i.e., groups that weakly decrease reaction rate), “moderatelydeactivating groups” (i.e., groups that moderately decrease reactionrate), and “strongly deactivating groups” (i.e., groups that stronglydecrease reaction rate).

Non-limiting examples of “weakly activating groups” are alkyl groups(e.g., methyl, ethyl, and the like), aryl groups (e.g., phenyl,naphthyl, and the like), and unsaturated hydrocarbon moieties (e.g.,alkenyl, alkynyl, and the like). Non-limiting examples of “moderatelyactivating groups” are N-attached amides (—NHCOR) and O-attached esters(—OCOR). Non-limiting examples of “strongly activating groups” are —NH₂,—NHR, —NR₂, —OR (e.g., —OMe, -OEt, and the like), and —OH. Non-limitingexamples of “weakly deactivating groups” are halogen groups (e.g., —F,—Cl, —Br, and the like). Non-limiting examples of “moderatelydeactivating groups” are formyl (e.g., —CHO), ketones (—COR), carboxylicacid (—COOH), C-attached carboxylic esters (—COOR), carboxylic acidhalides (e.g., —COCl, and the like), and C-attached amides (—CONH₂,—CONHR, —CONHR₂, and the like). Non-limiting examples of “stronglydeactivating groups” are trihaloalkyl moieties (e.g., —CF₃, and thelike), —CN, S-attached sulfonates (e.g., —SO₃R, and the like),quaternary ammonium salts (e.g., —NH₃, ⁺—NR₃ ⁺, and the like), and —NO₂.

In certain aspects, the invention is based upon the surprising discoverythat fusion proteins comprising a cytochrome P450 enzyme and a reductaseenzyme can transfer a NO₂ functional group to the indole ring ofL-tryptophan with high regio-selectivity. Therefore, in some aspects,the disclosure provides a fusion protein comprising (i) a cytochromeP450 enzyme which catalyzes transfer of a nitro functional group to aL-tryptophan having at least one substitution on its indole ring; (ii)an amino acid linker; and, (iii) a catalytic domain of a reductaseenzyme. In some aspects, the fusion protein comprises an amino acidlinker that joins the reductase enzyme to a terminus of the cytochromeP450 enzyme. In some embodiments, the reductase is joined to theC-terminus of the cytochrome P450 enzyme.

In certain aspects, the invention is based upon the surprising discoverythat fusion proteins comprising a cytochrome P450 enzyme and a reductaseenzyme can transfer a NO₂ functional group to the indole ring ofL-tryptophan with high regio-selectivity. Therefore, in some aspects,the disclosure provides a fusion protein comprising (i) a cytochromeP450 enzyme which catalyzes transfer of a nitro functional group to acompound of Formulae Ia-IXa; (ii) an amino acid linker; and, (iii) acatalytic domain of a reductase enzyme. In some aspects, the fusionprotein comprises an amino acid linker that joins the reductase enzymeto a terminus of the cytochrome P450 enzyme. In some embodiments, thereductase is joined to the C-terminus of the cytochrome P450 enzyme.

Cytochrome P450 enzymes (CYPs) form a super-family of heme-thiolatecontaining enzymes. CYP enzymes regio/stereo-selectively catalyze avariety of chemical reactions and generally require the consumption of areducing agent, for example NADPH. Effectivly transferring electronsfrom the reducing agent to the heme center requires a proper interactionbetween the CYP and suitable auxiliary redox proteins. Based on thetypes of redox proteins required for activity, CYPs typically areorganized into three classes (class I, class II and class III). Thecatalytic activity of class I CYPs depends on both a redoxin protein,such as ferredoxins (Fer), and a reductase enzyme, such as flavinadenine dinucleotide (FAD)-containing reductase (Frd) enzymes.Non-limiting examples of class I CYPs include but are not limited toCYP1A1, CYP2A6, CYP3A5, CYP11A1, CYP101, CYP105 and CYP107A1 and TxtE.TxtE is a cytochrome P450 enzyme naturally found in Streptomyces scabiesthat transfers a nitro group (NO₂) to thaxtomin phytotoxins. The naturalsubstrate of TxtE is L-tryptophan. As a class I CYP, catalytic activityof TxtE normally requires the interaction with a small redox 2Fe-2Siron-sulfur ferodoxin and FAD reductase.

Class II and class III CYPs are self-sufficient enzymes, in which theheme domains are fused with reductase domains as single polypeptides (DeMot and Parret 2002). As used herein, the term “self-sufficient enzyme”refers to a cytochrome P450 enzyme linked to a reductase catalyticdomain, which does not require the activity of any auxiliary redoxprotein (e.g. a ferredoxin or reductase enzyme) other than the reductasedomain linked to said cytochrome P450 enzyme in order to perform itsintended function. Examples of naturally occurring self-sufficientcytochrome P450 enzymes include but are not limited to CYP505A1,CYP102A1 (P450BM3), P450 PFOR, and P450RhF.

The skilled artisan recognizes that additional examples of class I, IIand III CYPs may be identified using methods generally known in the art,for example mining a database of CYP sequences, such as disclosed byNelson, DR (2009) The Cytochrome P450 Homepage. Human Genomics 4, 59-65.

In some aspects, the invention relates to artificial, or non-naturallyoccurring self-sufficient cytochrome P450 enzymes. As used herein, theterm “artificial cytochrome P450 enzyme” refers to a non-naturallyoccurring fusion protein comprising a non-self-sufficient cytochromeP450 enzyme and a catalytic domain of a reductase enzyme. Withoutwishing to be bound by any particular theory, the fusion of a reductasedomain to a naturally non-self-sufficient cytochrome P450 enzyme confersself-sufficient function to the P450 enzyme yet maintains the functionalcharacteristics of the P450 enzyme. For example, a class I cytochromeP450 enzyme fused to a reductase domain does not require the activity ofauxiliary redox proteins in order to transfer a nitro group to asubstrate. Therefore, in some embodiments, the self-sufficientcytochrome P450 enzyme is a class I cytochrome P450 enzyme.

In some aspects, the disclosure relates to the transfer of a nitro group(NO₂) to an aromatic molecule comprising an indole ring. In someembodiments, the aromatic molecule is L-tryptophan. In some embodiments,the aromatic molecule is substituted. In some embodiments, the aromaticmolecule is substituted on its indole ring. In some embodiments, thesubstituted aromatic molecule is substituted L-tryptophan. Varioussubstitutions on the indole ring of L-tryptophan are contemplatedherein. For example, the indole ring of L-tryptophan may comprise one ormore substititons at carbon 4, 5, 6, and/or 7. In embodiments, one ofcarbon 4 and carbon 7 is not substituted when used as a startingmaterial. The substitution may be halogen (e.g. F, C1, Br, I),substituted or unsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃), substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), provided that the substitution does notcomprise a “moderately deactivating group”, a “strongly deactivatinggroup”, or a group that does not sterically hinder interaction betweenthe P450 enzyme and the reductase enzyme catalytic domain. Sterichindrance may occur if the substitution on the indole ring comprises alarge molecule that impedes access of the substrate to the active siteof P450 enzyme or prevents interaction of reductase with P450 enzyme.

In some aspects, the disclosure relates to the transfer of a nitro group(NO₂) to an aromatic molecule comprising an indole ring. In someembodiments, the aromatic molecule is L-tryptophan. In some embodiments,the aromatic molecule is substituted. In some embodiments, the aromaticmolecule is substituted on its indole ring. In some embodiments, thearomatic molecule is substituted on the benzoid portion of an indolemoiety (i.e., at the 4-, 5-, 6-, or 7-position). In some embodiments,the substituted aromatic molecule is a compound of Formulae Ia-IXa. Thesubstitution may be halogen (e.g. F, Cl, Br, I), substituted orunsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃), substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala), provided that the substitution does not comprise a“moderately deactivating group”, a “strongly deactivating group”, ordoes not sterically hinder interaction between the P450 enzyme and thereductase enzyme catalytic domain. As used herein, the terms “moderatelydeactivating group” and “strongly deactivating group” refer to afunctional moiety that moderately or strongly reduces the rate ofelectrophilic aromatic substitution (e.g., nitration), respectively,relative to the corresponding unsubstituted aromatic moiety, as iswell-known in the art. Non-limiting examples of “moderately deactivatinggroups” are formyl (e.g., —CHO), ketones, carboxylic acid (—COOH),C-attached carboxylic esters, carboxylic acid halides (e.g., —COCl, andthe like), and C-attached amides. Non-limiting examples of “stronglydeactivating groups” are trihaloalkyl moieties (e.g., —CF₃, and thelike), —CN, S-attached sulfonates, quaternary ammonium salts, and —NO₂.Steric hindrance may occur if the substitution on the indole ringcomprises a large molecule that impedes access of the substrate to theactive site of P450 enzyme or prevents interaction of reductase withP450 enzyme.

In certain aspects, each of X¹, X², X³, Y¹, Y², and Y³ is independently—H, a “weakly deactivating group”, a “weakly activating group”, a“moderately activating group”, or a “strongly activating group”.Non-limiting examples of “weakly activating groups” are alkyl groups(e.g., methyl, ethyl, and the like), aryl groups (e.g., phenyl,naphthyl, and the like), and unsaturated hydrocarbon moieties (e.g.,alkenyl, alkynyl, and the like). Non-limiting examples of “moderatelyactivating groups” are N-attached amides and O-attached esters.Non-limiting examples of “strongly activating groups” are —NH₂,secondary amines, tertiary amines, alkoxy (e.g., —OMe, -OEt, and thelike), and —OH. Non-limiting examples of “weakly deactivating groups”are halogen groups (e.g., —F, —Cl, —Br, and the like).

Some aspects of the invention relate to the inventors' recognition andappreciation that cytochrome P450 TxtE transfers a nitro group tosubstituted L-tryptophan. Accordingly, in some embodiments, thecytochrome P450 enzyme of the fusion protein is a TxtE enzyme. As usedherein, the term “TxtE enzyme” refers to a (i) polypeptide comprisingthe entire amino acid sequence of TxtE, (ii) a portion of TxtE whichmaintains the function of catalyzing transfer of a nitro functionalgroup to a L-tryptophan having at least one substitution on its indolering, or (iii) an enzyme which catalyzes transfer of a nitro functionalgroup to a L-tryptophan having at least one substitution on its indolering and is at least 95% homologous to the amino acid sequence of TxtE.The skilled artisan recognizes that for a portion of TxtE to maintainthe nitration function, the portion must include active site residues ofTxtE, for example Arg59, Asn293, Thr296 and Glu394. However, geneticmodification of residues at a location of the TxtE polypetide remotefrom the active site may maintain the activity of the enzyme. As usedherein, the term “genetic modification” refers to amino acidsubstitution (conservative, missense and/or non-sense), deletion and/orinsertion. Thus in some embodiments, a portion of TxtE comprises atleast 1, at least 2, at least 3, at least 4, at least 5, at least 10, atleast 20, at least 30, at least 40, at least 50, at least 75, or atleast 100 genetic modifications relative to wild-type TxtE. In someembodiments a portion of TxtE is truncated relative to wild-type TxtE.Truncations may occur at the N-terminus or C-terminus of the portion ofTxtE. For example, a portion of TxtE may be truncated by 1, 2, 3, 4, 5,10, 20, 30, 40, 50, 75, 100 or 200 amino acids at it N-terminus orC-terminus relative to wild-type TxtE. Methods of genetically modifyingTxtE or portions thereof and screening for retention of functionalactivity are known in the art and available to the skilled artisan. Forexample, TxtE may be modified by directed evolution or randommutagenesis and biochemcially assayed for the capability to transfer anitro group to L-tryptophan having at least one substitution on itsindole ring. In some embodiments, a TxtE enzyme may be an enzyme whichcatalyzes transfer of a nitro functional group to a L-tryptophan havingat least one substitution on its indole ring and has less than 95%homologous to the amino acid sequence of TxtE. In some embodiments, theenzyme has about 90%, about 80%, about 70%, about 60% or about 50%homology to the amino acid sequence of TxtE.

Some aspects of the invention relate to the inventors' recognition andappreciation that cytochrome P450 TxtE transfers a nitro group to acompound of Formulae Ia-IXa. Accordingly, in some embodiments, thecytochrome P450 enzyme of the fusion protein is a TxtE enzyme. As usedherein, the term “TxtE enzyme” refers to a (i) polypeptide comprisingthe entire amino acid sequence of TxtE, (ii) a portion of TxtE whichmaintains the function of catalyzing transfer of a nitro functionalgroup to a compound of Formulae Ia-IXa, or (iii) an enzyme whichcatalyzes transfer of a nitro functional group to compound of FormulaeIa-IXa and is at least 95% homologous to the amino acid sequence ofTxtE. The skilled artisan recognizes that for a portion of TxtE tomaintain the nitration function, the portion must include active siteresidues of TxtE, for example Arg59, Asn293, Thr296 and Glu394. However,genetic modification of residues at a location of the TxtE polypetideremote from the active site may maintain the activity of the enzyme. Asused herein, the term “genetic modification” refers to amino acidsubstitution (conservative, missense and/or non-sense), deletion and/orinsertion. Thus in some embodiments, a portion of TxtE comprises atleast 1, at least 2, at least 3, at least 4, at least 5, at least 10, atleast, at least 30, at least 40, at least 50, at least 75, or at least100 genetic modifications relative to wild-type TxtE. In someembodiments a portion of TxtE is truncated relative to wild-type TxtE.Truncations may occur at the N-terminus or C-terminus of the portion ofTxtE. For example, a portion of TxtE may be truncated by 1, 2, 3, 4, 5,10, 20, 30, 40, 50, 75, 100 or 200 amino acids at it N-terminus orC-terminus relative to wild-type TxtE. Methods of genetically modifyingTxtE or portions thereof and screening for retention of functionalactivity are known in the art and available to the skilled artisan. Forexample, TxtE may be modified by directed evolution or randommutagenesis and biochemcially assayed for the capability to transfer anitro group to a compound of Formulae Ia-IXa. In some embodiments, aTxtE enzyme may be an enzyme which catalyzes transfer of a nitrofunctional group to a compound of Formulae Ia-IXa and has less than 95%homologous to the amino acid sequence of TxtE. In some embodiments, theenzyme has about 90%, about 80%, about 70%, about 60% or about 50%homology to the amino acid sequence of TxtE.

In some aspects, the disclosure provides fusion proteins comprising acatalytic domain of a reductase enzyme. As used herein, the term“reductase enzyme” refers to an enzyme that catalyzes a reductionreaction. Non-limiting examples of reductase enzymes include thioredoxinreductase, cytochrome P450 reductase and flavin adenine dinucleotide(FAD) reductase. In some embodiments, the reductase enzyme is aprokaryotic reductase enzyme. In some embodiments, the reductase enzymeis a bacterial reducatase enzyme. In some embodiments, the bacterialreductase enzyme naturally occurs in a self-sufficient cytochrome P450,for example CYP102A1 (P450BM3) reductase or a P450RhF reductase.

In some embodiments, the fusion protein comprises an amino acid linker.As used herein, the term “linker” refers to an amino acid sequence thatjoins two larger polypeptide domains to form a single fusionpolypeptide. Amino acid linkers are well known to those skilled in theart and include flexible linkers (e.g. glycine rich linkers such as[GGGS]n where n>2), rigid linkers (e.g. poly-proline rich linkers) andcleavable linkers (e.g. photocleavable and enzyme-sensitive linkers). Insome embodiments, the amino acid linker joins a catalytic domain of areductase enzyme to a termunus of a cytochrome P450 enzyme. As usedherein, the term “terminus” refers to the ends of a polypeptide sequencerelative to the start codon of said polypeptide. For example, theN-terminus of a polypeptide is the end of the polypeptide containing thestart codon (AUG) of the polypeptide, whereas the C-terminus of thepolypeptide is the end of the polypeptide opposite of the start codon.In some embodiments, the amino acid linker joins the a catalytic domainof a reductase enzyme to the C-termunus of a cytochrome P450 enzyme. Insome embodiments, the amino acid linker joins CYP102A1 (P450BM3)reductase or P450RhF reductase to the C-terminus of a TxtE enzyme.

The length of amino acid linkers is also contemplated by the disclosure.Amino acid linker length is known to affect the folding and orientationof fusion polypeptides. For example, a linker that is too long canprevent the interaction of a reductase domain with the cytochrome P450enzyme to which it is linked. (It is also known that long linkers canfold and take on specific orientations that can be desireable.)Conversely, a linker that is too short can cause a reductase enzyme tosterically inhibit binding of substrate to the active site of the P450enzyme to which it is linked. Generally, linkers may range in lengthfrom about 5 to about 30 amino acids. In some embodiments, linkers rangefrom 6 to 20 amino acids in length. In some embodiments, linkers rangefrom 6 to 16 amino acids or from 10 to 16 amino acids in length. In someembodiments, linkers range from 6 to 10 amino acids in length. In someembodiments, the length of the linker is 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acids in length.

In some aspects, the disclosure relates to an expression constructcomprising a fusion protein as described by the disclosure. As usedherein, the term “expression construct” refers to an artificiallyconstructed molecule comprising a nucleic acid (e.g. DNA) capable ofartificially carrying foreign genetic material into another cell (forexample, a bacterial cell). In some embodiments, vectors carry commonfunctional elements including an origin of replication, a multicloningsite, a selectable marker and optionally a promoter sequence. In someembodiments, the selectable marker is a bacterial resistance gene, forexample kanamycin, chloramphenicol or P3-lactamase. Non-limitingexamples of vectors include plasmids, viral vectors, cosmids, andartificial chromosomes. In some embodiments, the vector is a high-copyplasmid. In some embodiments, the vector is a low-copy plasmid. In someembodiments, the vectors of the disclosure are maintained inside cells.In some embodiments, the vectors of the disclosure are maintained in anon-cellular environment, for example as part of a kit. Methods ofintroducing vectors into bacteria are well known in the art anddescribed, for example, in Current Protocols in Molecular Biology,Ausubel et al. (Eds), John Wiley and Sons, New York, 2007.

In some aspects, the disclosure relates to isolated nucleic acidsencoding the fusion proteins described herein. As used herein “nucleicacid” refers to a DNA or RNA molecule. Nucleic acids are polymericmacromolecules comprising a plurality of nucleotides. In someembodiments, the nucleotides are deoxyribonucleotides orribonucleotides. In some embodiments, the nucleotides comprising thenucleic acid are selected from the group consisting of adenine, guanine,cytosine, thymine, uracil and inosine. In some embodiments, thenucleotides comprising the nucleic acid are modified nucleotides.Non-limiting examples of natural nucleic acids include genomic DNA andplasmid DNA. In some embodiments, the nucleic acids of the instantdisclosure are synthetic. As used herein, the term “synthetic nucleicacid” refers to a nucleic acid molecule that is constructed via thejoining nucleotides by a synthetic or non-natural method. Onenon-limiting example of a synthetic method is solid-phaseoligonucleotide synthesis. In some embodiments, the nucleic acids of theinstant disclosure are isolated.

In some aspects, the disclosure relates to compounds produced byaromatic nitration. Certain aspects of the disclosure relate tounnatural compounds produced by the transfer of a nitro group toL-tryptophan by a cytochrome P450 enzyme. Accordingly, in some aspectsthe disclosure provides a method for producing a compound of:

wherein in Formula I, X¹ is halogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₂₋₆ alkenyl, substituted orunsubstituted C₂₋₆ alkynyl, substituted or unsubstituted, monocyclic,3-to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic,3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl,substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl,—OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein R^(Ala) is independently hydrogen, substituted or unsubstitutedacyl, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring andwherein the Ala of —OR^(Ala) is not H;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala),

wherein R^(Ala) is independently hydrogen, substituted or unsubstitutedacyl, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring andwherein the Ala of —OR^(Ala) is not H,

and Y is NO₂.

In some aspects, the disclosure relates to compounds produced byaromatic nitration. Certain aspects of the disclosure relate tounnatural compounds produced by the transfer of a nitro group toL-tryptophan or an L-tryptophan derivative (e.g., a compound of FormulaeIa-IXa) by a cytochrome P450 enzyme. Accordingly, in some aspects thedisclosure provides a method for producing a compound of Formula I, or apharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof,the method comprising contacting a compound of Formula Ia:

with:

-   -   (i) at least one reductase enzyme; and    -   (ii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula Ia, in the        presence of NAD(P)H;

to produce a compound of Formula I:

wherein:

each X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl, substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula IV, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IVa:

with:

-   -   (iii) at least one reductase enzyme; and    -   (iv) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula Ia, in the        presence of NAD(P)H;

to produce a compound of Formula IV:

wherein:

each X¹ is independently halogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₂₋₆ alkenyl, substituted orunsubstituted C₂₋₆ alkynyl, substituted or unsubstituted, monocyclic,3-to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic,3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl,substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl,—OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula V, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula Va:

with:

-   -   (iii) at least one reductase enzyme; and    -   (iv) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula Ia, in the        presence of NAD(P)H;

to produce a compound of Formula V:

wherein:

each X¹ is independently halogen, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstituted C₂₋₆ alkenyl, substituted orunsubstituted C₂₋₆ alkynyl, substituted or unsubstituted, monocyclic,3-to 6-membered carbocyclyl, substituted or unsubstituted, monocyclic,3- to 6-membered heterocyclyl, substituted or unsubstituted phenyl,substituted or unsubstituted, monocyclic, 5- to 6-membered heteroaryl,—OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

each of X² and X³ is, independently, hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala);

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

Y is NO₂;

R₁ is H or optionally substituted alkyl;

R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl. In another aspect, R₁ is H or alkyl.In another aspect, R₁ is H. In another aspect, R₁ is alkyl. In anotheraspect, R₁ is H methyl. In another aspect, R₂ is H. In another aspect,R₁ and R₂ are each H. In another aspect, R₁ is alkyl and R₂ is H. Inanother aspect, R₁ is methyl and R₂ is H.

In some embodiments, X² and X³ are hydrogen.

In some aspects the disclosure provides a method for producing acompound of:

wherein in Formula II each of Y¹, Y², and Y³ is, independently,hydrogen, halogen, substituted or unsubstituted C₁₋₆ alkyl, substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala),

wherein R^(Ala) is independently hydrogen, substituted or unsubstitutedacyl, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring andwherein the Ala of —OR^(Ala) is not H;

and X is NO₂, provided that at least one of Y¹, Y², and Y³ is nothydrogen.

In another aspect, the disclosure relates to a method of producing acompound of Formula II, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IIa:

with:

-   -   (v) at least one reductase enzyme; and    -   (vi) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula II:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In another aspect, the disclosure related to a method of producing acompound of Formula VI, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula VIa:

with:

-   -   (v) at least one reductase enzyme; and    -   (vi) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula VI:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

provided that at least one of Y¹, Y², and Y³ is not hydrogen.

In another aspect, the disclosure related to a method of producing acompound of Formula VII, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula VIIa:

with:

-   -   (vii) at least one reductase enzyme; and    -   (viii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula VII:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

R₁ is H or optionally substituted alkyl;

R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl;

provided that at least one of Y¹, Y², and Y³ is not hydrogen. In anotheraspect, R₁ is H or alkyl. In another aspect, R₁ is H. In another aspect,R₁ is alkyl. In another aspect, R₁ is H methyl.

In another aspect, R₂ is H. In another aspect, R₁ and R₂ are each H. Inanother aspect, R₁ is alkyl and R₂ is H. In another aspect, R₁ is methyland R₂ is H.

In another aspect, the disclosure relates to a method of producing acompound of Formula III, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IIIa:

with:

-   -   (iii) at least one reductase enzyme; and    -   (iv) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIIa, in the        presence of NAD(P)H;

to produce a compound of Formula III:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula VIII, or a pharmaceutically acceptable salt,prodrug, hydrate, or solvate thereof, the method comprising contacting acompound of Formula VIIIa:

with:

-   -   (vii) at least one reductase enzyme; and    -   (viii) a cytochrome P450 enzyme which catalyzes transfer of a        nitro functional group to a compound of Formula IIa, in the        presence of NAD(P)H;

to produce a compound of Formula VIII:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring.

In another aspect, the disclosure related to a method of producing acompound of Formula IX, or a pharmaceutically acceptable salt, prodrug,hydrate, or solvate thereof, the method comprising contacting a compoundof Formula IXa:

with:

-   -   (ix) at least one reductase enzyme; and    -   (x) a cytochrome P450 enzyme which catalyzes transfer of a nitro        functional group to a compound of Formula IIa, in the presence        of NAD(P)H;

to produce a compound of Formula IX:

wherein:

each of Y¹, Y², and Y³ is, independently, hydrogen, halogen, substitutedor unsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala); and

wherein each R^(Ala) is independently hydrogen, substituted orunsubstituted acyl, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, or asulfur protecting group when attached to a sulfur atom, or two instancesof R^(Ala) are joined to form a substituted or unsubstituted,heterocyclic ring, or substituted or unsubstituted, heteroaryl ring;

X is NO₂;

R₁ is H or optionally substituted alkyl;

R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl. In another aspect, R₁ is H or alkyl.In another aspect, R₁ is H. In another aspect, R₁ is alkyl. In anotheraspect, R₁ is H methyl. In another aspect, R₂ is H. In another aspect,R₁ and R₂ are each H. In another aspect, R₁ is alkyl and R₂ is H. Inanother aspect, R₁ is methyl and R₂ is H.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆.

The term “aliphatic” includes both saturated and unsaturated, straightchain (i.e., unbranched), branched, acyclic, cyclic, or polycyclicaliphatic hydrocarbons, which are optionally substituted with one ormore functional groups. As will be appreciated by one of ordinary skillin the art, “aliphatic” is intended herein to include, but is notlimited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, andcycloalkynyl moieties. Thus, the term “alkyl” includes straight,branched and cyclic alkyl groups. An analogous convention applies toother generic terms such as “alkenyl”, “alkynyl”, and the like.Furthermore, the terms “alkyl”, “alkenyl”, “alkynyl”, and the likeencompass both substituted and unsubstituted groups. In certainembodiments, “lower alkyl” is used to indicate those alkyl groups(cyclic, acyclic, substituted, unsubstituted, branched or unbranched)having 1-6 carbon atoms. In general, alkyl, alkenyl, and alkynyl groupscontain 1-20 aliphatic carbon atoms. In embodiments, the alkyl, alkenyl,and alkynyl groups employed in the invention contain 1-6 aliphaticcarbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynylgroups employed in the invention contain 1-4 carbon atoms. Illustrativealiphatic groups thus include, but are not limited to, for example,methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂-cyclopropyl,vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl,—CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl,cyclopentyl, —CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,—CH₂-cyclohexyl moieties and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

“Alkyl” in general refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀alkyl”). In embodiments, an alkyl group has 1 to 6 carbon atoms (“C₁₋₆alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms(“C₁₋₅ alkyl”). In some embodiments, an alkyl group has 1 to 4 carbonatoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl group has 1 to 3carbon atoms (“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1to 2 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl grouphas 1 carbon atom (“C₁ alkyl”). In some embodiments, an alkyl group has2 to 6 carbon atoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groupsinclude methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl(C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), and n-hexyl (C₆). Additional examples of alkylgroups include n-heptyl (C₇), n-octyl (C₈) and the like. Unlessotherwise specified, each instance of an alkyl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents(e.g., halogen, such as F). In certain embodiments, the alkyl group isunsubstituted C₁₋₁₀ alkyl (e.g., —CH₃ (Me), unsubstituted ethyl (Et),unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr),unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g.,unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu ort-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)).In certain embodiments, the alkyl group is substituted C₁₋₁₀ alkyl (suchas substituted C₁₋₆ alkyl, e.g., —CF₃, Bn).

“Alkenyl”, in general, refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds, and no triple bonds (“C₂₋₂₀ alkenyl”). Inembodiments, an alkenyl group has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”).In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C₂₋₅alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms(“C₂₋₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon doublebonds can be internal (such as in 2-butenyl) or terminal (such as in1-butenyl). Examples of C₂₋₄ alkenyl groups include ethenyl (C₂),1-propenyl (C₃), 2-propenyl (C₃), 1- butenyl (C₄), 2-butenyl (C₄),butadienyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄ alkenyl groups as well as pentenyl (C₅),pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples ofalkenyl include heptenyl (C₇), octenyl (C₅), octatrienyl (C₅), and thelike. Unless otherwise specified, each instance of an alkenyl group isindependently optionally substituted, i.e., unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) withone or more substituents. In certain embodiments, the alkenyl group isunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis substituted C₂₋₁₀ alkenyl. In an alkenyl group, a C═C double bond forwhich the stereochemistry is not specified (e.g., —CH═CHCH₃ or

may be an (E)- or (Z)-double bond.

“Alkynyl”, in general, refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds, and optionally one or more double bonds(“C₂₋₂₀ alkynyl”). In embodiments, an alkynyl group has 2 to 6 carbonatoms (“C₂₋₆ alkynyl”). In some embodiments, an alkynyl group has 2 to 5carbon atoms (“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has2 to 4 carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynylgroup has 2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, analkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or morecarbon-carbon triple bonds can be internal (such as in 2-butynyl) orterminal (such as in 1-butynyl). Examples of C₂₋₄ alkynyl groupsinclude, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl(C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkynyl groups as well aspentynyl (C₅), hexynyl (C₆), and the like. Additional examples ofalkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”)or substituted (a “substituted alkynyl”) with one or more substituents.In certain embodiments, the alkynyl group is unsubstituted C₂₋₁₀alkynyl. In certain embodiments, the alkynyl group is substituted C₂₋₁₀alkynyl.

“Carbocyclyl” or “carbocyclic”, in general, refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbonatoms (“C₃₋₁₀ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In embodiments, a carbocyclyl group has 3 to 6 ring carbonatoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclyl group has5 to 10 ring carbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆carbocyclyl groups include, without limitation, cyclopropyl (C₃),cyclopropenyl (C₃), cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl(C₅), cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆),cyclohexadienyl (C₆), and the like.

Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclic ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclic ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. Unless otherwise specified, each instance of acarbocyclyl group is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is unsubstituted C₃₋₁₀ cycloalkyl. Incertain embodiments, the cycloalkyl group is substituted C₃₋₁₀cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 memberedheterocyclyl”). In heterocyclyl groups that contain one or more nitrogenatoms, the point of attachment can be a carbon or nitrogen atom, asvalency permits. A heterocyclyl group can either be monocyclic(“monocyclic heterocyclyl”) or a fused, bridged, or spiro ring system,such as a bicyclic system (“bicyclic heterocyclyl”), and can besaturated or can be partially unsaturated. Heterocyclyl bicyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclic ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclicring, or ring systems wherein the heterocyclic ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclic ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclic ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. In certainembodiments, the heterocyclyl group is unsubstituted 3-10 memberedheterocyclyl. In certain embodiments, the heterocyclyl group issubstituted 3-10 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8membered non-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-6 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-6 membered heterocyclyl”). In some embodiments, the 5-6 memberedheterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen,and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2ring heteroatoms selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has one ring heteroatomselected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing one heteroatominclude, without limitation, azetidinyl, oxetanyl and thietanyl.Exemplary 5-membered heterocyclyl groups containing one heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanyl.Exemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pielectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₋₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. Unlessotherwise specified, each instance of an aryl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

“Aralkyl” is a subset of alkyl and aryl and refers to an optionallysubstituted alkyl group substituted by an optionally substituted arylgroup. In certain embodiments, the aralkyl is optionally substitutedbenzyl. In certain embodiments, the aralkyl is benzyl. In certainembodiments, the aralkyl is optionally substituted phenethyl. In certainembodiments, the aralkyl is phenethyl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 pi electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, i.e., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

A heteroaryl group is a 5-10 membered aromatic ring system having ringcarbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-10 membered heteroaryl”). A heteroaryl group canbe a 5-8 membered aromatic ring system having ring carbon atoms and 1-4ring heteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group isa 5-6 membered aromatic ring system having ring carbon atoms and 1-4ring heteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-6 membered heteroaryl”). In some embodiments, the 5-6 memberedheteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, andsulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ringheteroatoms selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selectedfrom nitrogen, oxygen, and sulfur. Unless otherwise specified, eachinstance of a heteroaryl group is independently optionally substituted,i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a“substituted heteroaryl”) with one or more substituents. In certainembodiments, the heteroaryl group is unsubstituted 5-14 memberedheteroaryl. In certain embodiments, the heteroaryl group is substituted5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl, and thiophenyl.Exemplary 5-membered heteroaryl groups containing two heteroatomsinclude, without limitation, imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroarylgroups containing three heteroatoms include, without limitation,triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-memberedheteroaryl groups containing four heteroatoms include, withoutlimitation, tetrazolyl. Exemplary 6-membered heteroaryl groupscontaining one heteroatom include, without limitation, pyridinyl.Exemplary 6-membered heteroaryl groups containing two heteroatomsinclude, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.Exemplary 6-membered heteroaryl groups containing three or fourheteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing oneheteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

“Heteroaralkyl” is a subset of alkyl and heteroaryl and refers to anoptionally substituted alkyl group substituted by an optionallysubstituted heteroaryl group.

“Unsaturated” or “partially unsaturated” refers to a group that includesat least one double or triple bond. A “partially unsaturated” ringsystem is further intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl groups) as herein defined. Likewise, “saturated” refers toa group that does not contain a double or triple bond, i.e., containsall single bonds.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, which are divalent bridging groups, are further referred tousing the suffix -ene, e.g., alkylene, alkenylene, alkynylene,carbocyclylene, heterocyclylene, arylene, and heteroarylene.

An atom, moiety, or group described herein may be unsubstituted orsubstituted, as valency permits, unless otherwise provided expressly.The term “optionally substituted” refers to substituted orunsubstituted.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups are optionally substituted (e.g., “substituted” or“unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety. In certain embodiments, the substituent isa carbon atom substituent. In certain embodiments, the substituent is anitrogen atom substituent. In certain embodiments, the substituent is anoxygen atom substituent. In certain embodiments, the substituent is asulfur atom substituent.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR, —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃+X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; or two geminalhydrogens on a carbon atom are replaced with the group ═O, ═S,═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

-   -   each instance of R^(aa) is, independently, selected from C₁₋₁₀        alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀        carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14        membered heteroaryl, or two R^(aa) groups are joined to form a        3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,        wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,        aryl, and heteroaryl is independently substituted with 0, 1, 2,        3, 4, or 5 R^(dd) groups;    -   each instance of R^(bb) is, independently, selected from        hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),        —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))OR^(aa),        —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R, —SO₂OR^(cc),        —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),        —P(═O)₂R^(aa), —P(═O)(R^(a))₂, —P(═O)₂N(R^(cc))₂,        —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered        heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two        R^(bb) groups are joined to form a 3-14 membered heterocyclyl or        5-14 membered heteroaryl ring, wherein each alkyl, alkenyl,        alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is        independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)        groups;    -   each instance of R^(cc) is, independently, selected from        hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀        alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄        aryl, and 5-14 membered heteroaryl, or two R^(cc) groups are        joined to form a 3-14 membered heterocyclyl or 5-14 membered        heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,        carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;    -   each instance of R^(dd) is, independently, selected from        halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee),        —ON(R^(ff))₂, —N(R^(ff))₂, —N(R^(ff))₃+X, —N(OR^(ee))R^(ff),        —SH, —SR^(ee), —SSR^(ee), —C(═O)R^(ee), —CO₂H, —CO₂R^(ee),        —OC(═O)R^(ee), —OCO₂R^(ee), —C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂,        —NR^(ff)C(═O)R^(ee), —NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂,        —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee),        —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂,        —NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee),        —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂R^(ee), —OSO₂R^(ee),        —S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,        —C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),        —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀        carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10        membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,        carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two        geminal R^(dd) substituents can be joined to form ═O or ═S;    -   each instance of R^(ee) is, independently, selected from C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀        carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, and 3-10        membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,        carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently        substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; each        instance of R^(f) is, independently, selected from hydrogen,        C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀        carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10        membered heteroaryl, or two R^(ff) groups are joined to form a        3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,        wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,        aryl, and heteroaryl is independently substituted with 0, 1, 2,        3, 4, or 5 R^(gg) groups; and    -   each instance of R^(gg) is, independently, halogen, —CN, —NO₂,        —N₃, —SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆        alkyl)₂, —N(C₁₋₆ alkyl)₃+X, —NH(C₁₋₆ alkyl)₂+X, —NH₂(C₁₋₆        alkyl)+X⁻, —NH₃+X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆        alkyl), —NH(OH), —SH, —SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆        alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆        alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl),        —NHC(═O)(C₁ 6 alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁ 6 alkyl),        —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆        alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆ alkyl),        —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆        alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆        alkyl), —OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂,        —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl),        —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl,        —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆        alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂, —C(═O)S(C₁₋₆ alkyl),        —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆ alkyl),        —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆        alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered        heterocyclyl, 5-10 membered heteroaryl; or two geminal R^(gg)        substituents can be joined to form ═O or ═S; wherein X⁻ is a        counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), BF₄ ⁻, PF₄ ⁻, PF₆ ⁻, AsF₆ ⁻, SbF₆ ⁻,B[3,5-(CF₃)₂C₆H₃]₄]⁻, BPh₄ ⁻, Al(OC(CF₃)₃)₄ ⁻, carborane anions (e.g.,CB₁₁H₁₂ ⁻ or (HCB₁₁Me₅Br₆)⁻), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

“Acyl” refers to a moiety selected from the group consisting of—C(═O)R^(aa), —CHO, —CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa),—C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), or —C(═S)SR^(aa), wherein R^(aa) andR^(bb) are as defined herein.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substituents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(a))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc), and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc),vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate(Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys). Incertain embodiments, a nitrogen protecting group described herein is Bn,Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts.

Exemplary oxygen atom substituents include, but are not limited to,—R^(aa), —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R′)₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. In certain embodiments, the oxygen atom substituent present onan oxygen atom is an oxygen protecting group (also referred to as ahydroxyl protecting group). Oxygen protecting groups are well known inthe art and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, JohnWiley & Sons, 1999, incorporated herein by reference. Exemplary oxygenprotecting groups include, but are not limited to, methyl,t-butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl(MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl,4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM),2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP),3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl,4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,4-methoxytetrahydrothiopyranyl S,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). In certain embodiments, an oxygen protecting group describedherein is silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn,allyl, acetyl, pivaloyl, or benzoyl.

Exemplary sulfur atom substituents include, but are not limited to, —R,—C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. In certain embodiments, the sulfur atom substituent present on asulfur atom is a sulfur protecting group (also referred to as a thiolprotecting group). Sulfur protecting groups are well known in the artand include those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, incorporated herein by reference. In certain embodiments, asulfur protecting group described herein is acetamidomethyl, t-Bu,3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl.

In some aspects, the compound disclosure relates to a compound ofFormula I, wherein X¹ is halogen. Examples of halogens include F, Cl,Br, and I. In certain embodiments, the halogen is fluorine. FluorinatedL-tryptophan is a non-specific cytotoxic agent that acts as anantibiotic. In some embodiments, the L-tryptophan is fluorinated atposition 4 of the indole ring and nitrated at position 7 of the indolering. In some aspects, the compound disclosure relates to a compound ofFormula II, wherein Y¹, Y² or Y³ is halogen. In certain embodiments, thehalogen is fluorine. In some embodiments, the L-tryptophan isfluorinated at position 5, 6 or 7 of the indole ring and nitrated atposition 4 of the indole ring.

In some aspects, the compound disclosure relates to a compound ofFormula I, IV, or V, wherein at least one of X¹, X², or X³ is a weaklydeactivating group, a weakly activating group, a moderately activatinggroup, or a strongly activating group.

In other aspects, at least one of X¹, X², or X³ is H, halogen (e.g. F,Cl, Br, I), substituted or unsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃),substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala),

In another aspect, X₁ is halogen (e.g. F, Cl, Br, I), substituted orunsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃), substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala); and X² and X³ are each independently H, halogen (e.g. F, Cl,Br, I), substituted or unsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃),substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala). In another aspect, X¹ is halogen or C₁₋₆alkyl (e.g. methyl, CH₃). In another aspect, X¹ is halogen. In anotheraspect, X¹ is C₁₋₆ alkyl (e.g. methyl, CH₃). In another aspect, X¹ ishalogen or C₁₋₆ alkyl (e.g. methyl, CH₃) and at least one of X² and X³is hydrogen. In another aspect, X¹ is halogen and each of X² and X³ ishydrogen. In another aspect, X¹ if fluorine and each of X² and X³ ishydrogen. In another aspect, X¹ is C₁₋₆ alkyl and each of X² and X³ ishydrogen. In another aspect, X¹ is methyl and each of X² and X³ ishydrogen.

In some aspects, the compound disclosure relates to a compound ofFormula II, III, VI, VII, VIII, or IX, wherein each of Y¹, Y² or Y³ isindependently H, halogen (e.g. F, Cl, Br, I), substituted orunsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃), substituted orunsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆ alkynyl,substituted or unsubstituted, monocyclic, 3-to 6-membered carbocyclyl,substituted or unsubstituted, monocyclic, 3- to 6-membered heterocyclyl,substituted or unsubstituted phenyl, substituted or unsubstituted,monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala), —N(R^(Ala))₂, or—SR^(Ala). In another aspect, at least one of Y¹, Y² or Y³ is halogen orC₁₋₆ alkyl (e.g. methyl, CH₃). In another aspect, Y³ is halogen (e.g. F,Cl, Br, I), substituted or unsubstituted C₁₋₆ alkyl (e.g. methyl, CH₃),substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala); and Y¹ and Y² are each independently H,halogen (e.g. F, Cl, Br, I), substituted or unsubstituted C₁₋₆ alkyl(e.g. methyl, CH₃), substituted or unsubstituted C₂₋₆ alkenyl,substituted or unsubstituted C₂₋₆ alkynyl, substituted or unsubstituted,monocyclic, 3-to 6-membered carbocyclyl, substituted or unsubstituted,monocyclic, 3- to 6-membered heterocyclyl, substituted or unsubstitutedphenyl, substituted or unsubstituted, monocyclic, 5- to 6-memberedheteroaryl, —OR^(Ala), —N(R^(Ala))₂, or —SR^(Ala). In another aspect, Y³is halogen or C₁₋₆ alkyl (e.g. methyl, CH₃). In another aspect, Y³ ishalogen or C₁₋₆ alkyl (e.g. methyl, CH₃) and at least one of Y¹ and Y²is hydrogen. In another aspect, Y³ is halogen or C₁₋₆ alkyl (e.g.methyl, CH₃) and Y¹ and Y² are each hydrogen. In another aspect, Y³ ishalogen. In another aspect, Y³ is halogen and at least one of Y¹ and Y²is hydrogen. In another aspect, Y³ is halogen and Y¹ and Y² are eachhydrogen. In certain embodiments, Y³ is fluorine and at least one of Y¹and Y² is hydrogen. In another aspect, Y³ is fluorine and Y¹ and Y² areeach hydrogen. In another aspect, Y³ is C₁₋₆ alkyl. In another aspect,Y³ is C₁₋₆ alkyl and at least one of Y¹ and Y² is hydrogen. In anotheraspect, Y³ is C₁₋₆ alkyl and Y¹ and Y² are each hydrogen. In certainembodiments, Y³ is methyl and at least one of Y¹ and Y² is hydrogen. Inanother aspect, Y³ is methyl and Y¹ and Y² are each hydrogen. Thedisclosure also relates to pharmaceutical compositions comprising acompound of Formula I or a compound of Formula II and a pharmaceuticallyacceptable carrier. As used herein the term “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions. Pharmaceuticalcompositions can be prepared as described below. The active ingredientsmay be admixed or compounded with any conventional, pharmaceuticallyacceptable carrier or excipient. The compositions may be sterile.

The disclosure also relates to pharmaceutical compositions comprising acompound of Formulae I-IX, or a pharmaceutically acceptable salt,prodrug, hydrate, or solvate thereof, and a pharmaceutically acceptablecarrier. As used herein the term “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. Pharmaceutical compositions can beprepared as described below. The active ingredients may be admixed orcompounded with any conventional, pharmaceutically acceptable carrier orexcipient. The compositions may be sterile.

A carrier is said to be a “pharmaceutically acceptable carrier” if itsadministration can be tolerated by a recipient patient. Sterilephosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known in the art.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

It will be understood by those skilled in the art that any mode ofadministration, vehicle or carrier conventionally employed and which isinert with respect to the active agent may be utilized for preparing andadministering the pharmaceutical compositions of the present invention.Illustrative of such methods, vehicles and carriers are those described,for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970), thedisclosure of which is incorporated herein by reference. Those skilledin the art, having been exposed to the principles of the invention, willexperience no difficulty in determining suitable and appropriatevehicles, excipients and carriers or in compounding the activeingredients therewith to form the pharmaceutical compositions of theinvention.

In some embodiments, a compound of Formula I or Formula II isincorporated into a polypeptide. For example, nitration of anL-typtophan having a substitution on its indole ring results information of a compound of Formula I or Formula II. It is known in theart that L-tryptophan and its derivatives may be incorporated intopolypeptides to form artificial or unnatural proteins, for example asdisclosed by Methods in Molecular Biology, vol. 32: Protein EngineeringProtocols, Amdt and MUller (Eds.), Humana Press, N J, 2007.

In some embodiments, a compound of Formulae I-IX is incorporated into apolypeptide. For example, nitration of a compound of Formulae Ia-IXaresults in formation of a compound of Formulae I-IX. It is known in theart that L-tryptophan and its derivatives (e.g., compounds of FormulaeI-IX) may be incorporated into polypeptides to form artificial orunnatural proteins, for example as disclosed by Methods in MolecularBiology, vol. 32: Protein Engineering Protocols, Amdt and Müller (Eds.),Humana Press, N J, 2007.

In some aspects, the disclosure relates to methods for producing acompound of Formula I or Formula II. In some embodiments, the methodcomprises contacting an L-tryptophan having at least one substitution onits indole ring with at least one reductase enzyme and a cytochrome P450enzyme which catalyzes transfer of a nitro functional group to anL-tryptophan having at least one substitution on its indole ring in thepresence of NADPH. The skilled artisan appreciates that the L-tryptophanmay have substitution on any member of the indole ring. For example, theL-tryptophan may have substitution at position 4, 5, 6 or 7 of theindole ring. In some embodiments, the substitution is a halogensubstitution. In some embodiments, the halogen substitution is afluorine substitution. The method may utilize a native cytchrome P450enzyme and associated redox proteins or a fusion protein. For example,the L-tryptophan may be contacted with wild-type TxtE cytochrome P450enzyme, ferredoxin and ferredoxin reductase in the presence of NAD(P)Hto produce a compound having Formula I or Formula II. In someembodiments, the L-tryptophan is contacted with a fusion protein, forexample a TxtE enzyme terminally-linked to a catalytic domain of areductase enzyme, in the presence of NAD(P)H to produce a compoundhaving Formula I or Formula II.

In some aspects, the disclosure relates to methods for producing acompound of Formulae I-IX, or a pharmaceutically acceptable salt,prodrug, hydrate, or solvate thereof. In some embodiments, the methodcomprises contacting a compound of Formulae Ia-IXa with at least onereductase enzyme and a cytochrome P450 enzyme which catalyzes transferof a nitro functional group to a compound of Formulae Ia-IXa in thepresence of NADPH. The skilled artisan appreciates that the compound ofFormulae Ia-IXa may have substitution on any member of the indole ring.For example, the compound of Formulae Ia-IXa may have substitution atposition 4, 5, 6 or 7 of the indole ring. In some embodiments, thesubstitution is halogen. In some embodiments, the halogen is fluorine.The method may utilize a native cytchrome P450 enzyme and associatedredox proteins or a fusion protein. For example, the compound ofFormulae Ia-IXa may be contacted with wild-type TxtE cytochrome P450enzyme, ferredoxin and ferredoxin reductase in the presence of NAD(P)Hto produce a compound having Formulae I-IX. In some embodiments, thecompound of Formulae Ia-IXa is contacted with a fusion protein, forexample a TxtE enzyme terminally-linked to a catalytic domain of areductase enzyme, in the presence of NAD(P)H to produce a compoundhaving Formulae I-IX.

The invention also relates, in some aspects, to a method for producing adi-substituted nitrated indole. In some aspects, the method comprisescontacting an L-tryptophan molecule having a singly-substituted indolering, in the presence of NAD(P)H, with at least one reductase enzyme anda cytochrome P450 enzyme that catalyzes transfer of a nitro functionalgroup to an L-tryptophan having at least one substitution on its indolering. In aspects the L-tryptophan having at least one substitution onits indole ring is substituted with other than a nitrate. In aspects theL-tryptophan molecule having at least one substitution on its indolering is singly-substituted on its indole ring and the resultingnitro-substituted L-tryptophan is a di-substituted nitrated indole. Inaspects the method further comprises isolating the nitratedL-tryptophan. In aspects the method further comprises isolating thedi-substituted nitrated indole portion of the L-tryptophan molecule fromthe L-tryptophan molecule. Methods of removing or isolating indole ringsare known in the art. For example, the enzyme tryptophanase may be usedto deaminate tryptophan to produce an indole ring.

The invention also relates, in some aspects, to a method for producing adi-substituted nitro-substituted indole. In some aspects, the methodcomprises contacting a compound of Formulae Ia-IXa wherein X², and X³ inFormula Ia, IVa, or Va are both hydrogen or one of Y¹, Y², Or Y³ inFormulae IIa, IIIa, VIa, VIIa, VIIIa, or IXa is not hydrogen, in thepresence of NAD(P)H, with at least one reductase enzyme and a cytochromeP450 enzyme that catalyzes transfer of a nitro functional group to acompound of Formulae Ia-IXa. In aspects the compound of Formulae Ia-IXais substituted with a substituent other than a nitro group. In aspectsY³in Formulae IIa, IIIa, VIa, VIIa, VIIIa, or IXa is hydrogen. In aspectsthe method further comprises isolating the compound of Formulae I-IX. Inaspects the method further comprises isolating the indole portion of thecompound of Formulae I-IX from the compound of Formulae I-IX. Methods ofremoving or isolating indole rings are known in the art. For example,the enzyme tryptophanase may be used to deaminate tryptophan to producean indole ring.

In another aspect, the invention is directed to tryptophan or anytryptophan derivitave (e.g., compounds of Formulae I, Ia, II, IIa, III,IIIa, IV, IVa, V, Va, VI, VIa, VII, VIIa, VIII, VIIIa, IX, or IXa) andthe use of the aforementioned tryptophan or tryptophan derivatives inany of the processes or methods delineated herein. The tryptophanderivatives, Formulae Ia-IXa, can be prepared according to any syntheticmethods known in the art [e.g., Thomas Sorrell, Organic Chemistry,University Science Books, Sausalito, 1999; Smith and March, March'sAdvanced Organic Chemistry, 5^(th) Edition, John Wiley & Sons, Inc., NewYork, 2001; Larock, Comprehensive Organic Transformations, VCHPublishers, Inc., New York, 1989; and Carruthers, Some Modern Methods ofOrganic Synthesis, 3^(rd) Edition, Cambridge University Press,Cambridge, 1987]. For example, tryptophan derivatives, Formulae Ia-IXa,can be prepared from the corresponding indole, Formulae Ib-IVb, viareaction with serine and acetic acid/acetic anhydride, as outlined inBlaser, G. et al. (2008) Tetrahedron letters., 49 (17). pp. 2795-2798.Other chemical and enzymatic methods are also known for convertingindoles (e.g., compounds of Formulae Ib-IVb) to the correspondingtryptophan derivatives (e.g., compounds of Formulae Ia-IXa) [Eto et al.,Bull. Chem. Soc. Japan (1989), 62(3), pages 961-963; Li et al.,Tetrahedron (2014), 70(42), pages 7753-7762; Wartmann et al., Eur. J.Org. Chem. (2013), 2013(9), pages 1649-1652; Murai et al., J. Org. Chem.(2012), 77(19), pages 8581-8587; Mollica et al., Tet. Lett. (2011),52(20), pages 2583-2585; Heemstra et al., J. Am. Chem. Soc., (2008),130(43), pages 14024-14025; Yamada et al., Chem. Pharm. Bull. (2005),53(10), pages 1277-1290; Li et al., Tet. Lett. (2004), 45(46), pages8569-8573; Kim et al., Syn. Comm. (2004), 34(16), pages 2931-2943;Konda-Yamada et al., Tetrahedron (2002), 58(39), pages 7851-7861;WO2001094345; Zhang et al., Tet. Lett. (1995), 36(41), pages 7411-7314;Filler et al., Can. J. Chem. (1989), 67(11), pages 1837-1841; Ojima etal., J. Org. Chem. (1989), 54(19), pages 4511-4522; Schmidt et al.,Liebigs Annalen der Chemie (1985), 4, pages 785-793; Petrovic et al.,Amino Acids (2013), 44(5), pages 1329-1336; Frese et al., ChemCatChem(2014), 6(5), pages 1270-1276; Smith et al., Org. Lett. (2014), 16(10),pages 2622-2625].

Indoles of Formulae Ib-IVb can be purchased from commercial sources orcan be prepared by any methods known in the art for preparing and/ormodifying indoles. Non-limiting examples of such processes are Bartoliindole synthesis, Mannich reaction, Fischer indole synthesis, Nenitzescuindole synthesis, and the like.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting.

EXAMPLES Example 1: Materials and Methods

General Chemicals, DNA Sub-Cloning, and Bacterial Strains

Molecular biology reagents and enzymes were supplied by FisherScientific. Primers were ordered from Sigma-Aldrich. 4-F-dl-Tryptophanwas purchased from MP Biomedicals (Santa Ana, Calif.), while NOC-5 waspurchased from EMD Millipore. Other chemicals and solvents werepurchased from Fisher Scientific and Sigma-Aldrich. Escherichia coliDH5a (Life Technologies) was used for cloning and plasmid harvesting,while E. coli BL21-GOLD (DE3) (Agilent) was used for proteinoverexpression. E. coli strains were grown in Luria-Bertani broth orTerrific broth. Preparation and manipulation of plasmid DNA from E. coliwas accomplished following manufacture protocols from Thermo Scientificor Zymo Research. DNA sequencing was performed at Eurofins. A ShimadzuProminence UHPLC system (Kyoto, Japan) fitted with an Agilent Poroshell120 EC-C18 column (2.7 μm, 3.0×50 mm), coupled with a PDA detector wasused for HPLC analysis. A 3200 QTRAP (Applied Biosystems) equipped witha Shimadzu UPLC system was used for LC-MS/MS analysis in the studies.All NMR spectra were recorded in 50 mM DCl on an Agilent 600 MHzspectrometer using a 1.5 mm High Temperature Superconductor Probe in theAMRIS facility at the University of Florida. The instrument was operatedat 600.17 MHz for ¹H and 150.9 MHz for ¹³C. Spectroscopy data werecollected using VNMRJ Version-4.0. HRMS data were obtained using anAgilent LC-TOF mass spectrometer equipped with electrospray sourcedetector.

Construction of Self-Sufficient TxtE Variants

TxtE gene was amplified from S. scabies 87.22 genomic DNA using a pairof TxtEFN and TxtERH primers (Table 1) in PCR reaction. The PCR mixture(50 μL) contained 50 ng template, 2 μM of each primer, 0.1 mM of dNTP,3% dimethyl sulfoxide, and 0.5 μl Phusion high fidelity DNA polymerasein 1×GC reaction buffer. Reaction conditions consisted of an initialdenaturation step at 98° C. for 30 s followed by 30 cycles of 98° C. for10 s, 70° C. for 20 s, and 72° C. for 30 s, and a final extension of 72°C. for 5 min. The PCR product was analyzed by agarose gel and extractedwith a GeneJET Gel Extraction Kit (Thermo) following a manufacture'sprotocol. To create the TxtE-P450BM3 reductase (BM3R) domain fusiongene, TxtE gene was amplified using a pair of TxtEFN and TxtEBRR primerswhile TxtEBRF and BRRS primers were used to amplify BM3R gene, which wasthen followed by an overlapping PCR (Higuchi et al. 1988). Similarly,TxtE-RhFRed and TxtE-RhFRed* fusion genes were generated by fusing TxtEgene with P450RhF reductase domain (RhFRed) gene. Corresponding primerswere included in Table 1. Purified PCR products and pET28b were digestedwith the same sets of restriction enzymes and corresponding linear DNAswere ligated to generate expression constructs. All inserts in theconstructs were sequenced to exclude mutations introduced during PCRamplification and gene manipulation.

TABLE 1 Primers for production of fusion protein SEQ ID NameSequence (5′→3′) Function NO. TxtE-FN CACCCATGGTGACCGTCCCCTCGC TxtE   1cloning TxtE-RH ATATAAGCTTGCGGAGGCTGAGCG TxtE   2 GCAG cloning TxtEBRFGCCGCTCAGCCTCCGCTCTGCTAA TxtE-  3 AAAAGTACGC BM3R  fusion TxtEBRRGCGTACTTTTTTAGCAGAGCGGAG TxtE-  4 GCTGAGCGGC BM3R  fusion BRRSATCGAGCTCGACCCAGCCCACACG TxtE-  5 TCTTTTGC BM3R  fusion TxtERedFCCGCTCAGCCTCCGCGTGCTGCAC TxtE-  6 CGCCATC RhFRed  fusion TxtERedRGATGGCGGTGCAGCACGCGGAGGC TxtE-  7 TGAGCGG RhFRed  fusion TxtE8ARedFGCCGCTCAGCCTCCGCCATGTGCG TxtE-  8 ATTGGCGTC RhFRed*  fusion TxtE8ARedRGACGCCAATCGCACATGGCGGAGG TxtE-  9 CTGAGCGGC RhFRed*  fusion RedRHCTCAAGCTTGAGGCGCAGGGCCAG TxtE- 10 GCG RhFRed  fusion

Heterologous Expression and Purification of Recombinant Proteins

Insert-validated constructs were transformed into E. coli BL21(DE3)-GOLD competent cells for protein expression. Cells harboring theconstructs were cultured in Terrific Broth medium supplemented withkanamycin (50 μg/ml) and 1× trace metal solution (1000× stock solution:50 mM FeCl₃, 20 mM CaCl₂, 10 mM MnSO₄, 10 mM ZnSO₄, 2 mM CoSO₄, 2 mMCuCl₂, 2 mM NiCl₂, 2 mM Na₂MoO₄, and 2 mM H₃BO₃). Cultures were grown at37° C., 250 rpm until OD₆₀₀ reached 0.6. Protein expression was theninduced by isopropyl-β-D-thiogalactopyranoside (IPTG) with a finalconcentration of 0.1 mM. The cultures were further grown at 16° C., 250rpm for 16 hours. After centrifugation (5,000 g, 10 min, and 4° C.),cell pellets were stored in −80° C. or directly used for proteinpurification. For protein purification, cell pellets were firstresuspended in the suitable volumes of lysis buffer (cellbiomass:volume=1:4) [25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mMimidazole, 3 mM β-mercaptoethanol (BME) and 10% glycerol]. Solubleproteins were released by sonication. After centrifugation at 35,000×gat 4° C. for 30 min, the clear supernatants were incubated withpre-equilibrated Ni-NTA agarose resin (Thermo) at 4° C. for 2 h. Theresins were washed with 10 volumes of lysis buffer with 30 mM imidazole,and recombinant P450s were then eluted in lysis buffer with 50 to 320 mMimidazole. After SDS-PAGE analysis, elution solution fractionscontaining P450s were combined and concentrated. The proteins were thenexchanged into storage buffer (25 mM Tris-HCl, pH8.0, 100 mM NaCl, 3 mM(3ME, and 10% glycerol) using PD-10 column according to themanufacture's protocol, aliquoted and stored at −80° C. until needed.The concentrations of functional P450s were accurately measured by COdifference spectroscopy (Omura and Sato 1964).

Spectral Analysis of Self-Sufficient TxtE Variants

Purified TxtE and its fusion enzymes were spectrally analyzed followinga previous protocol (Ding et al. 2008). Briefly, the absorbance spectra(400-600 nm) of TxtE variants (3 μM) in Tris-HCl (25 mM, pH 8) bufferwere recorded with a Shimadzu UV2700 dual beam UV-Vis spectrophotometer.The ferric heme of enzymes was then saturated with carbon monoxide(Airgas) through bubbling and the spectra of the saturated enzymesolutions were recorded. Immediately, sodium dithionite solution (30 μL,0.5 M) was added to reduce ferric ion, and reduced spectra were takensubsequently. CO reduced difference spectra of all enzymes were createdby subtracting the CO binding spectra from the reduced spectra. Datawere further analyzed by GraphPad Prism 4. Substrate binding affinitiesto P450s were measured using 1.5 μM of enzyme solutions in 25 mMTris-HCl, pH 8.0. Not more than 10 μl of substrate stock solutionsprepared in the above buffer were added to the sample cuvette with aninterval of 0.5 al, and the spectra were recorded from 300 nm to 500 nmeach time. The equal volume of buffer was added to the referencecuvette. The changes in absorbance (ΔA) were determined by subtractingthe absorbance at ˜420 nm from that at ˜390 nm. Data were then fitted toMichaelis-Menten equation using GraphPad Prism 4.

Catalytic Activities of Self-Sufficient TxtE Variants

P450 reactions contained 0.5 mM substrate, 1 mM NADP⁺, 1 mM glucose, ˜10units/mL self-prepared glucose dehydrogenase crude extract, 1 mM NOC-5in 100 μL of Tris-HCl buffer (100 mM, pH 8.0). As the positive control,TxtE reaction was also re-constructed in the above mixture furthersupplemented with 0.43 μM spinach Fer and 0.33 μM Frd. The reactionswere initiated by adding 1.5 μM P450s, and incubated at 20° C., 300 rpmon a thermostat (Eppendorf) for 2 hours. Methanol (200 μl) was thenadded to stop the reactions. After centrifugation, 10 l solutions wereanalyzed by HPLC. The HPLC column kept at 40° C. was eluted first with1% solvent B (acetonitrile with 0.1% formic acid) for 0.5 min and thenwith a linear gradient of 1-20% solvent B in 2 min, followed by anotherlinear gradient of 20-99% solvent B in 0.5 min. The column was furthercleaned with 99% solvent B for 0.5 min and then re-equilibrated with 1%solvent B for 2 min. The flow rate was set as 1.5 mL/min, and theproducts were detected at 211 nm with a PDA detector. All enzymereactions were performed at least in duplicate.

Biochemical Characterization of Self-Sufficient TxtE Variants.

The stability of NO donor NOC-5 was first examined. Its solution wasincubated at different pH value (4.5 to 9.5) and temperatures (4 to 65°C.) for 30 min. It was then used as NO donor in the P450 nitrationreactions. NOC-5 was stable in all tested pH values but was decomposedquickly and significantly at temperatures higher than 25° C. Todetermine pH effects on the activity of TxtE and TxtEBM3R, enzyme (1.5μM) reactions were performed in 100 mM Tris-Cl or sodium phosphate withvarious pH values (4.5 to 9.5) at 20° C., 300 rpm for 30 min. Todetermine enzyme pH stability, 5 aL of 30 μM enzyme solutions wereincubated at buffers with different pH value (4.5 to 9.5). After 15minutes, other reaction components (95 aL) were mixed to initiatenitration reactions as described above. To test enzyme thermostability,TxtE and TxtEBM3R were incubated in 100 mM Tris-HCl (pH 8.0) atdifferent temperatures (4° C., 15° C., 20° C., 25° C., 30° C., 35° C.,40° C., 45° C., 55° C., and 65° C.) for 15 min. After cooling on ice for5 min, enzyme solutions were centrifuged and then used to initiatereactions at 20° C., 300 rpm for 30 min. Products were quantitated byHPLC as described above. Conversion rate (%) was calculated by theequation of the area of under the 4-nitro-l-tryptophan peak/the totalareas of both substrate and product peaks*100. All experiments wereperformed at least in duplicate. In this study, the T₅₀ is defined asthe temperature at which a 15-minute incubation of the enzyme causes theloss of one-half of the enzyme activity, relative to a 100% activityreference enzyme that does not undergo incubation.

Large-Scale Enzymatic Synthesis of Nitrated Fluoro-Tryptophan Analogs

To isolate sufficient amounts of nitrated fluoro-tryptophan analogs forstructural determination, 18 μM TxtEBM3R was used in a 10-mL reactionmixture containing 1.5 mM fluorinated substrate, 3 mM NADP⁺, 3 mMglucose, ˜30 units/mL self-prepared glucose dehydrogenase crude extract,3 mM NOC-5 in 100 mM Tris-HCl buffer (pH 8.0). The reactions in a 200-mlflask were incubated at 20° C., 250 rpm overnight, and then terminatedby 20 mL methanol or acidification to pH 1.0 with 6 M HCl. Aftercentrifugation, the supernatants were concentrated in vacuo and thenfreeze-dried. The powders were redissolved in 3 ml methanol.Semi-preparation was performed by HPLC (Shimazu) with a semi-prep C18column (Agilent ZORBAX SB-C18, 5 μm, 9.4×250 mm). The column kept at 40°C. was eluted first with 20% solvent B (acetonitrile with 0.1% formicacid) for 3 min and then with a linear gradient of 20-54% solvent B for3 min, followed by a linear gradient of 54-77% solvent B for 6 min. Thecolumn was then cleaned by 99% solvent B for 1 min and re-equilibratedwith 20% solvent B for 1 min. The flow rate was set as 3 mL/min, and theproducts were detected at 211 nm with a PDA detector. All isolates werecombined, concentrated, freeze-dried, and then weighed.

LC-MS/MS and NMR Analysis

A SHIMADZU Prominence UPLC system fitted with an Agilent Poroshell 120EC-C18 column (2.7 μm, 3.0×50 mm) coupled with a Linear Ion TrapQuadrupole LC/MS/MS Mass Spectrometer system was used in the studies.The column was eluted with 1% solvent B (acetonitrile with 0.1% formicacid) for 2 min and then with a linear gradient of 1-20% solvent B in 8min, followed by another linear gradient of 20-99% solvent B in 2.5 min.The column was then cleaned by 99% solvent B for 0.5 min andre-equilibrated with 1% solvent B for 2.5 min. The flow rate was 0.5mL/min. For MS detection, the turbo spray conditions were identical forall chemicals (curtain gas: 30 psi; ion spray voltage: 5500 V;temperature: 750° C.; ion source gas 1: 60 psi; ion source gas 2: 70psi). For MS/MS analysis, the collision energy was 20 eV. In NMRanalysis, chemical shifts were reported in parts per million (ppm)downfield from tetramethylsilane. Proton coupling patterns weredescribed as singlet (s), doublet (d), double doublet (dd), triplet (t),and multiplet (m). 5-F-4-nitro-l-tryptophan: ¹H NMR (600 MHz, 50 mM DCl)δ 7.52 (d, J=8.8 Hz, 1H), 7.35 (s, 1H), 6.95 (dd, J=10.2, 10.2 Hz, 1H),4.04-3.93 (m, 1H), 3.23 (dd, J=15.3, 5.6 Hz, 2H), 3.05 (dd, J=15.3, 8.4Hz, 2H); ¹³C NMR (151 MHz, 50 mM DCl) δ 171.29, 151.69, 150.03, 134.52,131.66, 129.49, 129.41, 118.41, 118.34, 117.89, 110.47, 110.30, 105.89,105.86, 72.01, 62.46, 59.31, 53.65, 27.09. HRMS (ESI⁺): calc. forC₁₁H₁₁FN₃O₄[M+H]+: 268.0728, found: 268.0728. 4-F-7-nitro-l-tryptophan:¹H NMR (600 MHz, 50 mM DCl) δ 7.80 (dd, J=8.1, 8.1 Hz, 1H), 7.29 (s,1H), 7.22 (d, J=9.1 Hz, 1H), 4.29-4.23 (m, 1H), 3.43 (dd, J=11.7, 6.5Hz, 2H), 3.32 (dd, J=15.2, 8.2 Hz, 1H); ¹³C NMR (151 MHz, 50 mM DCl) δ171.29, 152.41, 150.65, 142.34, 142.24, 128.59, 128.48, 119.38, 115.29,115.18, 108.40, 108.35, 72.00, 62.45, 59.30, 53.73, 38.70, 26.72.

HRMS (ESI⁻): calc. for C₁₁H₁₁FN₃O₄[M−H]⁻ 266.0583, found: 266.0577.

Example 2: Preparation of Self-Sufficient TxtE Variants

TxtE promotes a regio-selective nitration on the C4 of L-tryptophanindole ring using O₂ and NO as co-substrates and consuming NADPH(FIG. 1) (Barry et al. 2012). Since the native redox partners of TxtEremain unidentified, spinach Fer and Frd were used to support thereaction. Three artificial self-sufficient TxtE fusion enzymes,TxtE-BM3R, TxtE-RhFRed, and TxtE-RhFRed* were designed by appendingNADPH-dependent reductase domains of P450BM3 and of P450RhF to theC-terminus of TxtE. The linker of TxtE-BM3R was predicted from P450BM3using software Domcut (Suyama and Ohara 2003). Two other fusion enzymesutilizing linkers of different lengths were produced. TxtE-RhFRedcontains the native linker length of P450RhF, while TxtE-RhFRed* addseight additional residues to the native linker. All three fusion enzymeswere expressed in E. coli and purified to homogeneity with over 85%purity by a single nickel affinity chromatography (FIG. 2A). Allrecombinant proteins showed calculated molecular weights, 112 kDa forTxtE-BM3R and about 82 kDa for both TxtE-RhFRed and TxtE-RhFRed*, inSDS-PAGE analysis (FIG. 2A). To assess the functional folding ofrecombinant fusion proteins, UV/Vis spectroscopy was used to recordtheir absorption spectra (FIG. 2B). The CO-bound oxidized form andreduced form of these enzymes resembled similar features to wild typeTxtE and other bacterial CYPs. Soret peaks were shifted from around 419nm in the oxidized forms to around 449 nm in the reduced-CO differenceforms, indicating the proper folding of all fusion enzymes. Theconcentrations of functional heme-enzymes were accurately determined bythis spectral approach, following the previously published protocols(Omura and Sato 1964; Ding et al. 2008).

Example 3: Catalytic Activity of TxtE Fusion Enzymes

Next, the catalytic activities of all three fusion enzymes (TxtE-BM3R,TxtE-RhFRed, and TxtE-RhFRed*) were assessed along with NADPH, the NOdonor NOC-5 and L-tryptophan. As a control, wild type TxtE wasreconstructed with spinach Fer and Fdr. HPLC analysis of reactionmixtures revealed that all fusion enzymes enabled the L-tryptophannitration reaction to a different extent (FIG. 2C). TxtE-BM3R exhibiteda higher conversion (109%) than the control, while both TxtE-RhFRed andTxtE-RhFRed* only reached 13% and 16% of the conversion level of thecontrol, respectively. To examine the extent to which the fusionarrangement influenced the substrate-enzyme interaction, which mightinduce the observed variation of enzyme activity, the binding affinityof L-tryptophan toward all fusion enzymes was measured. TxtE-RhFRedshowed the highest binding affinity with the K_(d) value of 18.21±1.38μM, followed by TxtE-BM3R (K_(d)=20.83±0.35 μM) and TxtE-RhFRed*(K_(d)=24.34±1.21 μM). These values remained in the same range as wildtype TxtE (K_(d)=24.77±1.07 μM). Therefore, activity differences offusion enzymes may be originated from electron transfer efficiencybetween TxtE and reductase domains. Interestingly, no nitrated productwas detected by LC-MS analysis when TxtE was incubated with a standaloneBM3R (data not shown), indicating the necessity of covalently linkingtwo domains to promote the catalytically active electron transfer.

Example 4: Biochemical Characterization of TxtE and TxtE-BM3R

In this example, the thermostability of both TxtE and TxtE-BM3R (FIG.3A) was examined. These enzymes were incubated under differenttemperatures (4 to 65° C.) for 15 min and then used in L-tryptophannitration reaction at 20° C. Both enzymes showed a similar level ofthermostability with the T₅₀ of around 45° C. (FIG. 3A). Afterincubation at 65° C. for 15 min, their activity was completely lost,indicating irreversible conformation changes at high temperature. Next,the pH dependence of TxtE and TxtE-BM3R using NOC-5 as the NO donor wasexamined (FIG. 3B). This reagent is stable over a broad pH range. Bothenzymes remained <5% activity at buffers with pH below 7.0. TxtE-BM3Rshowed over 50% activity from pH 7.5 to pH 9.5 and its optimal pH rangewas 8.0 to 8.5. TxtE's activity depended on a narrower pH range, and itsoptimal activity preferred to pH 8.0. The extent to which the stabilityof both enzymes were affected by buffers with different pH values wasfurther examined. After being incubated in these buffers for 15 min,enzymes were then used to nitrate L-tryptophan at pH 8.0, 20° C. (FIG.5). HPLC analysis revealed that enzyme activities were only minimallyaffected by the incubation in different buffers. This result suggestedthat the pH dependence of enzyme activity (FIG. 3B) was not associatedwith the enzyme pH stability.

Example 5: Enzymatic Production of Fluorinated Nitro-Tryptophan Analogs

This example describes the use of TxtE-BM3R to produce nitro-tryptophananalogs. In this study, commercially available racemic 4-F-DL-tryptophanand 5-F-L-tryptophan were chosen as unnatural substrates becausefluorine substitution is a common strategy used by medicinal chemists togenerate drug molecules with improved properties (Ilardi et al. 2014).The binding of both substrates toward TxtE and TxtE-BM3R was studied(FIG. 6). Similar to L-tryptophan, the two fluorinated substratesinduced type I spectral changes in both enzyme solutions. Compared withTxtE, the binding affinities between these substrates and TxtE-BM3R wereabout 60% tighter, indicating the BM3R might facilitate substratebinding (FIG. 6). In previous studies, D-tryptophan was unable to inducespectral changes in TxtE solution (Barry et al. 2012; Dodani et al.2014). This observation may suggest that 4-F-L-tryptophan of the racemicmixture is the actual ligand bound to TxtE and TxtE-BM3R. With currentinaccessibility to optically pure 4-F-L-tryptophan, the totalconcentration of the racemic mixture to calculate K_(d) values, whichthus underestimated the accurate binding affinities. Nonetheless,compared with native substrate L-tryptophan (K_(d)=20.83±0.35 μM), thebinding affinities between TxtE-BM3R and 4-F-DL-tryptophan(K_(d)=189.20±11.14 μM) and 5-F-L-tryptophan (K_(d)=84.18±4.37 μM) werelowered by about 8 and 3 times, respectively, reflecting the bindinginterferences induced by the F-substitution at different positions.Next, the influences of the fluorination substitution on enzyme activitywere examined. Remarkably, both TxtE and TxtE-BM3R slightly preferred5-F-L-tryptophan over L-tryptophan (1.2:1) in the nitration reaction. Inaddition, although the C4 in 4-F-L-tryptophan is occupied by a Fsubstitution, both enzymes were able to nitrate this substrate ascharacterized by HPLC and LC-MS/MS analysis (FIGS. 4A-4C). The overallconversion rate was, however, only about 20% of L-tryptophan.

Example 6: Structural Characterization of Fluorinated Nitro-TryptophanAnalogs

Structural characterization of nitrated F-tryptophan products were firstperformed by LC-MS/MS (FIGS. 4A-4C). Nitrated 5-F-L-tryptophan wasfragmented in the same pattern as that of 4-nitro-L-tryptophan in MS2spectra (FIGS. 4A-4B). However, the C5-F substitution not only increasedthe m/z values of all corresponding ions by 18 Da but also affected thedistribution of different ions (FIGS. 4A-4B). The most abundant ion inthe MS2 spectrum of 4-nitro-L-tryptophan had the m/z values of 159.0. Itwas switched to 174.2 in the MS2 spectrum of nitrated 5-F-L-tryptophan,corresponding to the non-fluorinated ion of 156.2. The most abundant ionin the MS2 spectrum of nitrated product with 4-F-DL-tryptophan as thesubstrate had an m/z value of 209.0 (FIG. 4C). Importantly, its overallfragmentation pattern was notably different with that of nitrated5-F-L-tryptophan. Putative chemical structures of red-labeled ions inthese MS2 spectra are shown in FIG. 7. This result suggested that5-F-L-tryptophan may be nitrated at the same site, the C4, asL-tryptophan but the nitration site at 4-F-L-tryptophan as the potentialreal substrate in the racemic mixture is different.

To further elucidate the nitro position in nitrated products, largescale enzymatic reactions were performed. About 90% of 5-F-L-tryptophanwas nitrated and about 2 milligrams of the nitro product as a yellowpowder were purified by a semi-preparative HPLC. Similarly, less than0.2 milligrams of putative nitro-4-F-L-tryptophan as a light beige solidwas isolated. Both products carried a single nitro group, and theircorresponding exact masses were confirmed in HRMS analysis (FIGS.8A-8B). Isolated products were further structurally characterized by ¹Hand ¹³C and 2D NMR analysis (FIGS. 9A, 9B, 10A, 10B, 11A, 11B, 12A, 12B,and Table 2). Examining the NMR data demonstrated that the C4 and the C7of 5-F-L-tryptophan and 4-F-L-tryptophan, respectively, are nitrated inTxtE-BM3R reactions. From the ¹H NMR spectrum of the nitro5-F-L-tryptophan product (FIGS. 9A-9B), the large coupling constant(J=10.2 Hz) of the triplet-like peak at δ 6.95 ppm (C₆) suggested asingle vicinal coupling with the fluorine atom. Furthermore, aneighboring doublet peak at δ 7.52 ppm (C₇) with a coupling constantJ=8.8 Hz defined an ortho substitution pattern of the two aromaticprotons. The aforementioned multiplicity and coupling constantstherefore determined the C4 nitro substitution in the 5-F-L-tryptophansubstrate, which was further confirmed by HSQC and HMBC analysis (FIGS.11A, 11B, 12A, and 12B). From its ¹H NMR spectrum, a triplet-like peakat δ 7.80 ppm (C₅) displayed a doublet of doublet split with twoapproximately equal coupling constants of 8.1 Hz, suggesting a vicinalcoupling with the fluorine atom. An ortho substitution pattern of thetwo aromatic protons was further defined by a large coupling constant(J=9.1 Hz) of neighboring doublet peak at δ 7.52 ppm (C₆). Together, thenitro site was determined to be the C7 of 4-F-L-tryptophan, which wasfurther confirmed by HSQC and HMBC analysis (FIGS. 11A, 11B, 12A, and12B). These results therefore revealed TxtE as a versatile nitratingbiocatalyst with remarkable regio-selectivity and substrate promiscuity.

TABLE 2 ¹³C and ¹H NMR data for 5-fluoro-4-nitro-1-tryptophan and4-fluoro- 7-nitro-1-tryptophan (recorded in 50 mM DCl) 5-F-4-nitro-L-Trp4-F-7-nitro-L-Trp Atom δ_(C) ^(a), type δ_(H) ^(b) (J in Hz) δ_(C) ^(a),type δ_(H) ^(b) (J in Hz) 2 131.7, CH 7.35 s 128.5, CH 7.29 s 3 105.9, C108.3, C 3a 117.9, C 115.2, C 4 129.5, C 151.5, C 5 150.9, C 119.4, CH7.80 dd (8.1, 8.1) 6 110.4, CH 6.95 dd (10.2,10.2) 108.4, CH 7.22 d(9.1) 7 118.4, CH 7.52 d (8.8) 128.6, C 7a 134.5, C 142.3, C 1′ 171.3, C171.3, C 2′  53.7, CH 3.99 m  53.7, CH 4.26 m 3′  27.1, CH₂ 3.23 dd(15.3, 5.6)  26.7, CH₂ 3.43 dd (15.2, 6.5) 3.05 dd (15.3, 8.4) 3.32 dd(15.2, 8.2)

Example 7: Artificial Self-Sufficient Cytochrome p450 Enzymes

This example describes a direct nitration reaction on the L-tryptophanindole ring with O₂ and NO as co-substrates that is catalyzed by theenzyme TxtE (FIG. 13A).

General Chemicals, DNA Sub-Cloning, and Bacterial Strains

Molecular biology reagents and enzymes were supplied by FisherScientific. Primers were ordered from Sigma-Aldrich. 4-F-dl-Tryptophanwas purchased from MP Biomedicals (Santa Ana, Calif.), while NOC-5[3-(Aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene] was purchasedfrom EMD Millipore. Marfey's reagent was purchased from FisherScientific. Other chemicals and solvents were purchased from FisherScientific and Sigma-Aldrich. Escherichia coli DH5a (Life Technologies)was used for cloning and plasmid harvesting, while E. coli BL21-GOLD(DE3) (Agilent) was used for protein overexpression. E. coli strainswere grown in Luria-Bertani broth or Terrific broth. Preparation andmanipulation of plasmid DNA from E. coli was accomplished followingmanufacture protocols from Thermo Scientific or Zymo Research. DNAsequencing was performed at Eurofins. A Shimadzu Prominence UHPLC system(Kyoto, Japan) fitted with an Agilent Poroshell 120 EC-C18 column (2.7μm, 3.0×50 mm), coupled with a PDA detector was used for HPLC analysisand determination of chemical UV spectra. A 3200 QTRAP (AppliedBiosystems) equipped with a Shimadzu UPLC system was used for LC-MS/MSanalysis in the studies. All NMR spectra were recorded in D₂O on anAgilent 600 MHz spectrometer using a 1.5 mm High TemperatureSuperconductor Probe in the AMRIS facility at the University of Florida.The instrument was operated at 600.17 MHz for ¹H and 150.9 MHz for ¹³C.Spectroscopy data were collected using VNMRJ Version-4.0. HRMS data wereobtained using an Agilent LC-TOF mass spectrometer equipped withelectrospray source detector.

Construction of Self-Sufficient TxtE Variants

TxtE gene (Genbank: FN554889 REGION: 3613916 . . . 3615136) wasamplified from genomic DNA of S. scabies 87.22 (NRRL B-24449) using apair of TxtEFN and TxtERH primers (Table 1) in PCR reaction. The PCRmixture (50 μL) contained 50 ng template, 2 μM of each primer, 0.1 mM ofdNTP, 3% dimethyl sulfoxide, and 0.5 μl Phusion high fidelity DNApolymerase in 1×GC reaction buffer. Reaction conditions consisted of aninitial denaturation step at 98° C. for 30 s followed by 30 cycles of98° C. for 10 s, 70° C. for 20 s, and 72° C. for 30 s, and a finalextension of 72° C. for 5 min. The PCR product was analyzed by agarosegel and extracted with a GeneJET Gel Extraction Kit (Thermo) following amanufacture's protocol. To create the TxtE-P450BM3 reductase (BM3R)domain fusion gene, TxtE gene was amplified using a pair of TxtEFN andTxtEBRR primers while TxtEBRF and BRRS primers were used to amplify BM3Rgene (GenBank: J04832.1) from the genome of B. megaterium ATCC 14581,which was then followed by an overlapping PCR. Similarly, TxtE-RhFRedand TxtE-RhFRed* fusion genes were generated by fusing TxtE gene withP450RhF reductase domain (RhFRed) gene (GenBank: AF459424.1) amplifiedfrom the template of pET21b-RhFRED. Corresponding primers were includedin Table 1 Purified PCR products and pET28a were digested with the samesets of restriction enzymes and corresponding linear DNAs were ligatedto generate expression constructs. All inserts in the constructs weresequenced to exclude mutations introduced during PCR amplification andgene manipulation.

Heterologous Expression and Purification of Recombinant Proteins

Insert validated constructs were transformed into E. coli BL21(DE3)-GOLD competent cells for protein expression. Cells harboring theconstructs were cultured in Terrific Broth medium supplemented withkanamycin (50 μg/ml) and 1× trace metal solution (1000× stock solution:50 mM FeCl₃, 20 mM CaCl₂, 10 mM MnSO₄, 10 mM ZnSO₄, 2 mM CoSO₄, 2 mMCuCl₂, 2 mM NiCl₂, 2 mM Na₂MoO₄, and 2 mM H₃BO₃). Cultures were grown at37° C., 250 rpm until OD₆₀₀ reached 0.6. Protein expression was theninduced by isopropyl-P3-D-thiogalactopyranoside (IPTG) with a finalconcentration of 0.1 mM. The cultures were further grown at 16° C., 250rpm for 16 hours. After centrifugation (5,000 g, 10 min, and 4° C.),cell pellets were stored at −80° C. or directly used for proteinpurification. For protein purification, cell pellets were firstresuspended in the suitable volumes of lysis buffer (cellbiomass:volume=1:4) [25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mMimidazole, 3 mM β-mercaptoethanol (BME) and 10% glycerol]. Solubleproteins were released by sonication. After centrifugation at 35,000×gat 4° C. for 30 min, the clear supernatants were incubated withpre-equilibrated Ni-NTA agarose resin (Thermo) at 4° C. for 2 h. Theresins were washed with 10 volumes of lysis buffer with 30 mM imidazole,and recombinant P450s were then eluted in lysis buffer with 50 to 320 mMimidazole. After SDS-PAGE analysis, elution solution fractionscontaining P450s were combined and concentrated. The proteins were thenexchanged into storage buffer (25 mM Tris-HCl, pH8.0, 100 mM NaCl, 3 mM(3ME, and 10% glycerol) using a PD-10 column according to themanufacture's protocol, aliquoted and stored at −80° C. until needed.The concentrations of functional P450s were accurately measured by COdifference spectroscopy.

Spectral Analysis of Self-Sufficient TxtE Variants

Purified TxtE and its fusion enzymes were spectrally analyzed followinga previous protocol. Briefly, the absorbance spectra (400-600 nm) ofTxtE variants (3 μM) in Tris-HCl (25 mM, pH 8) buffer were recorded witha Shimadzu UV2700 dual beam UV-Vis spectrophotometer. The ferric heme ofenzymes was then saturated with carbon monoxide (Airgas) throughbubbling and the spectra of the saturated enzyme solutions wererecorded. Immediately, sodium dithionite solution (30 μL, 0.5 M) wasadded to reduce ferric ion, and reduced spectra were taken subsequently.CO reduced difference spectra of all enzymes were created by subtractingthe CO binding spectra from the reduced spectra. Data were furtheranalyzed by GraphPad Prism 4. Substrate binding affinities to P450s weremeasured using 1.5 μM of enzyme solutions in 25 mM Tris-HCl, pH 8.0. Notmore than 10 μl of substrate stock solutions prepared in the abovebuffer were added to the sample cuvette with an interval of 0.5 Cal, andthe spectra were recorded from 300 nm to 500 nm each time. The equalvolume of buffer was added to the reference cuvette. The changes inabsorbance (AA) were determined by subtracting the absorbance at ˜420 nmfrom that at ˜390 nm. Data were then fitted to the equation ofΔA=ΔA_(max)[L]/(K_(d)+[L]) using GraphPad Prism 4.

Analytical and Semi-Preparative HPLC Analysis

For analytical analysis, the HPLC column kept at 40° C. was eluted firstwith 1% solvent B (acetonitrile with 0.1% formic acid) for 0.5 min andthen with a linear gradient of 1-20% solvent B in 2 min, followed byanother linear gradient of 20-99% solvent B in 0.5 min. The solvent Awas water with 0.1% formic acid. The column was further cleaned with 99%solvent B for 0.5 min and then re-equilibrated with 1% solvent B for 2min. The flow rate was set as 1.5 mL/min, and the products were detectedat 211 nm with a PDA detector. All enzyme reactions were performed atleast in triplicate.

For semi-preparative analysis, the column kept at 40° C. was elutedfirst with 20% solvent B (acetonitrile with 0.1% formic acid) for 3 minand then with a linear gradient of 20-54% solvent B for 3 min, followedby a linear gradient of 54-77% solvent B for 6 min. The column was thencleaned by 99% solvent B for 1 min and re-equilibrated with 20% solventB for 1 min. The flow rate was set at 3 mL/min, and the products weredetected at 211 nm with a PDA detector. All isolates were combined,concentrated, freeze-dried, and then weighed.

LC-MS/MS and NMR Analysis of Isolated Products

A SHIMADZU Prominence UPLC system fitted with an Agilent Poroshell 120EC-C18 column (2.7 μm, 3.0×50 mm) coupled with a Linear Ion TrapQuadrupole LC/MS/MS Mass Spectrometer system was used in the studies.The column was eluted with 1% solvent B (acetonitrile with 0.1% formicacid) for 2 min and then with a linear gradient of 1-20% solvent B in 8min, followed by another linear gradient of 20-99% solvent B in 2.5 min.The column was then cleaned by 99% solvent B for 0.5 min andre-equilibrated with 1% solvent B for 2.5 min. The flow rate was 0.5mL/min. For MS detection, the turbo spray conditions were identical forall chemicals (curtain gas: 30 psi; ion spray voltage: 5500 V;temperature: 750° C.; ion source gas 1: 60 psi; ion source gas 2: 70psi). For MS/MS analysis, the collision energy was 20 eV. In NMRanalysis, chemical shifts were reported in parts per million (ppm)downfield from tetramethylsilane. Proton coupling patterns weredescribed as singlet (s), doublet (d), double doublet (dd), triplet (t),and multiplet (m). 5-F-4-nitro-l-tryptophan: ¹H NMR (600 MHz, D₂O) δ7.52 (d, J=8.8 Hz, 1H), 7.35 (s, 1H), 6.95 (t, J=10.2 Hz, 1H), 4.04-3.93(m, 1H), 3.23 (dd, J=15.3, 5.6 Hz, 2H), 3.05 (dd, J=15.3, 8.4 Hz, 2H);¹³C NMR (151 MHz, D₂O) δ 171.29, 151.69, 150.03, 134.52, 131.66, 129.49,129.41, 118.41, 118.34, 117.89, 110.47, 110.30, 105.89, 105.86, 72.01,62.46, 59.31, 53.65, 27.09. HRMS (ESI⁺): calc. for C₁₁H₁FN₃O₄ [M+H]+:268.0728, found: 268.0728. 4-F-7-nitro-l-tryptophan: ¹H NMR (600 MHz,D₂O) δ 7.80 (t, J=8.1 Hz, 1H), 7.29 (s, 1H), 7.22 (d, J=9.1 Hz, 1H),4.29-4.23 (m, 1H), 3.43 (dd, J=11.7, 6.5 Hz, 2H), 3.32 (dd, J=15.2, 8.2Hz, 1H); ¹³C NMR (151 MHz, D₂O) δ 171.29, 152.41, 150.65, 142.34,142.24, 128.59, 128.48, 119.38, 115.29, 115.18, 108.40, 108.35, 72.00,62.45, 59.30, 53.73, 38.70, 26.72. HRMS (ESI⁻): calc. forC₁₁H₁₁FN₃O₄[M−H]⁻ 266.0583, found: 266.0577.

Marfey's Derivatization

To determine the stereoisomer of 4-F-dl-tryptophan in the nitrationreaction, 1-tryptophan, 4-nitro-l-tryptophan, 5-F-l-tryptophan,5-F-4-nitro-l-tryptophan, 4-F-dl-tryptophan, and nitrated4-F-dl-tryptophan from enzyme reactions and in purified form werereacted with Marfey's reagent following the manufacture manual (ThermoScientific). Derivatized products were analyzed by LC-MS with A SHIMADZUProminence UPLC system fitted with a Waters SymmetryShield™ RP-C18column (3.5 μm, 4.6×100 mm) and a Linear Ion Trap Quadrupole LC/MS/MSMass Spectrometer system. The column was eluted with 90% solvent A (0.05M triethylammonium acetate, pH 3.0), 10% solvent B (acetonitrile) for 2min and then with a linear gradient of 10-50% solvent B in 60 min. Thecolumn was then cleaned by 50% solvent B for 5 min and re-equilibratedwith 10% solvent B for 2 min. The flow rate was 0.5 mL/min. For MSdetection, the turbo spray conditions were the same as described above.

Catalytic Activities of Self-Sufficient TxtE Variants

P450 reactions contained 0.5 mM substrate, 1 mM NADP⁺, 1 mM glucose, ˜10units/mL self-prepared glucose dehydrogenase crude extract, 1 mM NOC-5[3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene] in 100 μL ofTris-HCl buffer (100 mM, pH 8.0). As the positive control, the TxtEreaction was also re-constructed in the above mixture furthersupplemented with 0.43 μM spinach Fer and 0.33 μM Frd. The reactionswere initiated by adding 1.5 μM P450s, and incubated at 20° C., 300 rpmon a thermostat (Eppendorf) for 2 hours. Methanol (200 μl) was thenadded to stop the reactions. After centrifugation, 10 μl solutions wereanalyzed by HPLC. Total turnover number (TTN) was reported as nmolproduct per nmol P450. The 4-NO₂-l-tryptophan was synthesized in alarge-scale enzymatic reaction to establish a standard curve for productquantification. To determine the coupling efficiency, NADPH (2 mM)replaced the NADPH regeneration system (NADP⁺, glucose, glucosedehydrogenase crude extract) in the reaction mixture. NADPH consumptionin enzyme reactions was measured at 340 nm (c=6.22 mM-cm¹⁻) with aShimadzu UV2700 dual beam UV-Vis spectrophotometer. Non-enzymaticoxidation of NADPH was subtracted as the background. The quantity ofnitrated product was determined by HPLC analysis as described above.Coupling efficiency (%) was determined as product (nmol)/consumed NADPH(nmol)×100. All reactions were independently repeated at least threetimes.

Biochemical Characterization of Self-Sufficient TxtE Variants.

The stability of NO donor NOC-5 was first examined by incubating it insolutions of different pH values (4.5 to 9.5) and temperatures (4 to 65°C.) for 30 min. NOC-5 was stable at all tested pH values but decomposedquickly and significantly at temperatures higher than 25° C. It was thenused as the NO donor in the following experiments. To determine pHeffects on the activity of TxtE and TxtEBM3R, enzyme (1.5 μM) reactionswere performed in 100 mM Tris-C₁ or sodium phosphate at different pHvalues (4.5 to 9.5) at 20° C., 300 rpm for 30 min. To determine enzymepH stability, 5 μL of 30 μM enzyme solutions were incubated in bufferswith different pH values (4.5 to 9.5). After 15 min, other reactioncomponents (95 μL) were mixed to initiate nitration reactions asdescribed above. To test enzyme thermostability, TxtE and TxtEBM3R wereincubated in 100 mM Tris-HCl (pH 8.0) at different temperatures (4° C.,15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 55° C., and 65°C.) for 15 min. After cooling on ice for 5 min, enzyme solutions werecentrifuged and then used to initiate reactions at 20° C., 300 rpm for30 min. Products were quantitated by HPLC as described above. Peak areawas determined with software installed in the Shimadzu Prominence UHPLCsystem. Conversion rate (%) was calculated as product(nmol)/product+substrate (nmol)×100 based on the standard curves of Trpand 4-NO₂-Trp generated. All experiments were performed at least intriplicate. In this study, the T₅₀ is defined as the temperature atwhich a 15-min incubation of the enzyme causes the loss of one-half ofthe enzyme activity, relative to a 100% activity reference enzyme thatdoes not undergo incubation.

Large-Scale Enzymatic Synthesis of Nitrated Fluoro-Tryptophan Analogs

To isolate sufficient amounts of nitrated fluoro-tryptophan analogs forstructural determination, 18 μM TxtEBM3R was used in a 10-mL reactionmixture containing 1.5 mM fluorinated substrate, 3 mM NADP⁺, 3 mMglucose, ˜30 units/mL self-prepared glucose dehydrogenase crude extract,3 mM NOC-5 in 100 mM Tris-HCl buffer (pH 8.0). The reactions in a 200-mlflask were incubated at 20° C., 250 rpm overnight, and then terminatedby 20 mL methanol addition or acidification to pH 1.0 with 6 M HCl.After centrifugation, the supernatants were concentrated in vacuo andthen freeze-dried. The products were redissolved in 3 ml methanol.Semi-preparation was performed with a semi-prep C₁₈ column (AgilentZORBAX SB-C18, 5 μm, 9.4×250 mm).

Creation of Properly Folded Self-Sufficient TxtE Variants

TxtE promotes a regio-selective nitration on the C4 of the 1-tryptophanindole ring using 02 and NO as co-substrates and consuming NADPH (FIG.13A). Although the native redox partners of TxtE remain unidentified,spinach Fer and Frd were able to support the reaction. To use TxtE as abroadly applicable biocatalyst for aromatic nitration, three artificialself-sufficient TxtE fusion enzymes, TxtE-BM3R, TxtE-RhFRed, andTxtE-RhFRed*, were designed by appending NADPH-dependent reductasedomains of P450BM3 and of P450RhF to the C-terminus of TxtE. The linkerof TxtE-BM3R was predicted from P450BM3 using software Domcut. Due toproven effects of linker lengths on catalytic activities of RhFRedfusion enzymes, two fusion enzymes were created. TxtE-RhFRed containedthe native linker length, while TxtE-RhFRed* has eight additionalresidues: this design offered the highest activities in previousstudies. All fusion enzymes were expressed in E. coli and purified tohomogeneity with over 85% purity by a single nickel affinitychromatography (FIG. 14A). All recombinant proteins showed calculatedmolecular weights, 112 kD for TxtE-BM3R and about 82 kD for bothTxtE-RhFRed and TxtE-RhFRed*, in SDS-PAGE analysis (FIG. 14A). To assessthe functional folding of recombinant fusion proteins, UV/Visspectroscopy was used to record their absorption spectra (FIG. 14B). TheCO-bound oxidized form and reduced form of these enzymes resembledsimilar features to wild type TxtE and other bacterial CYPs. Soret peakswere shifted from around 419 nm in the oxidized forms to around 449 nmin the reduced-CO difference forms (dotted lines), indicating the properfolding of all fusion enzymes.

Catalytic Performances of Fusion Enzymes

Catalytic activities of all three fusion enzymes were assessed, with thecontrol of wild type TxtE coupled with spinach Fer and Frd. HPLCanalysis of reaction mixtures revealed that all fusion enzymes nitrated1-tryptophan to a different extent (FIG. 14C). After 2 hours, TxtE-BM3Rexhibited slightly higher conversion (18.1%) than the control (15.9%),while both TxtE-RhFRed and TxtE-RhFRed* only reached 2.1% and 2.8%,respectively (FIG. 15). In consistency with these observations, TTNvalues of both TxtE-RhFRed and TxtE-RhFRed* were less than 10, whileTxtE-BM3R catalyzed over 320 nitration cycles, similar to TxtE (Table3). To examine the extent to which the fusion arrangement influenced thesubstrate-enzyme interaction, which might induce the observed variationof enzyme performance, the binding of L-tryptophan toward all fusionenzymes was assessed and the Type I spectral changes were observed(FIGS. 16A-16B). The shift from 420 to 390 nm is expected when a P450substrate displaces the axial water ligand from the heme iron, whichchanges the heme's iron from its low-spin state to the high-spin state(FIG. 16A). Binding affinities were then determined by examining thedifferential UV-Visible spectra with various substrate concentrations(FIG. 16B). TxtE-RhFRed showed the highest binding affinity with theK_(d) value of 18.2±1.4 μM, followed by TxtE-BM3R (K_(d)=20.8±0.4 μM)and TxtE-RhFRed* (K_(d)=24.3±1.2 μM). These values remained in the samerange as wild type TxtE (K_(d)=24.8±1.1 μM), indicating that fusiondesign had a minimal effect on substrate binding. Coupling efficiencywas then determined in order to evaluate electron transfer compatibilityof each enzyme during the nitration reaction (Table 3). Couplingefficiency of the TxtE-BM3R was slightly (1.9%) lower than TxtE (2.4%)coupled with Fer and Frd. However, TxtE-RhFRed and TxtE-RhFRed* showed8- and 24-folds decreased coupling efficiency, respectively, incomparison with TxtE. Therefore, the fusion organization between TxtEand RhFRed impaired proper electron transfer. Nonetheless, TxtE-BM3R wascomparable with TxtE in term of catalytic performance.

Binding affinities and relative nitration conversions for L-tryptophanand several substituted tryptophan analogs were also assessed (FIGS.29A-29B). Substrates with changes on the amine or carboxylate moieties(e.g., L-tryptophanol; a-Me-Trp; indole-3-pyruvate) showed significantlyweakened interactions while compounbds with substitution on the indolering maintained binding affinity (FIG. 29A). Furthermore, the nitrationactivity of TxtE and TxtEBM3R was assessed. In addition to substrate,enzyme reactions typically contained NADP+, an NADPH regeneration system(glucose and glucose dehydrogenase, GDH), spinach ferredoxin (Fer) andferredoxin reductase (Frd) as redox partners, and3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazinyl]-1-propanamine(NOC-5) as an NO donor. Reverse-phase UHPLC coupled with a PDA detectorand liquid chromatography-mass spectrometry (LC-MS, ESI positive) wasemployed to detect the nitrated products (FIG. 29B).

Biochemical Characterization of TxtE and TxtE-BM3R

To investigate biochemical properties of nitration biocatalysts,thermostability of both TxtE and TxtE-BM3R was examined (FIGS. 17A-17B).These enzymes were incubated at different temperatures (4 to 65° C.) for15 min and then used in the L-tryptophan nitration reaction at 20° C.Both enzymes showed a similar level of thermostability with a T₅₀ ofaround 45° C. (FIG. 17A). After incubation at 65° C. for 15 min, theiractivity was completely lost, indicating irreversible conformationalchanges at high temperature. Next, the pH dependence of TxtE andTxtE-BM3R was examined using NOC-5 as the NO donor (FIG. 17B). Thisreagent is stable over a broad pH range (data not shown). Both enzymesremained <5% activity in buffers with pH below 7.0. TxtE-BM3R showedover 50% activity from pH 7.5 to pH 9.5 and an optimal pH range of 8.0to 8.5. TxtE's activity depended on a narrower pH range with optimalactivity at pH 8.0. The extent to which the stability of both enzymeswere affected by buffers with different pH values was examined. Afterincubation in these buffers for 15 min, enzymes were then used tonitrate 1-tryptophan at pH 8.0, 20° C. (FIG. 18). HPLC analysis revealedthat enzyme activities were only minimally affected by the incubation indifferent buffers. This result suggested that the pH dependence ofenzyme activity (FIG. 17B) was not associated with enzyme pH stability.

Enzymatic Production of Fluorinated Nitro-Tryptophan Analogs

To expand the applications of TxtE-BM3R in nitration, the enzyme wasused to nitrate two unnatural substrates, commercially available racemic4-F-dl-tryptophan and 5-F-1-tryptophan. Fluorine substitution is acommon strategy used by medicinal chemists to generate drug moleculeswith improved properties. The two fluorinated substrates induced type Ispectral changes in solutions of TxtE and TxtE-BM3R (FIGS. 16A-16B).Similarly, they bound to TxtE-RhFRed and TxtE-RhFRed*. The bindingaffinity between each substrate and TxtE-BM3R was about 60% higher thanto TxtE, indicating that BM3R might facilitate substrate binding (FIG.19). In previous studies, d-tryptophan is unable to induce spectralchanges in TxtE solution.

Next, derivatized 1-tryptophan, 5-F-l-tryptophan, 4-F-dl-tryptophan, andtheir nitrated products were produced in the enzyme reactions and alsoproduced in purified form with Marfey's reagent. LC-MS analysisidentified only one derivatized product from nitrated 4-F-dl-tryptophanand suggested to be nitro-4-F-l-tryptophan (FIGS. 20A-20C). With currentinaccessibility to optically pure 4-F-l-tryptophan, the totalconcentration of the racemic mixture was used to calculate K_(d) values,which thus underestimated the accurate binding affinities. Nonetheless,compared with the native substrate 1-tryptophan (K_(d)=20.8±0.4 μM), thebinding affinities between TxtE-BM3R and 4-F-dl-tryptophan(K_(d)=189.2±11.1 μM) and 5-F-l-tryptophan (K_(d)=84.2±4.4 μM) werelowered by about 8 and 3 times, respectively, reflecting the bindinginterferences induced by the F-substitution. Next, the influences of thefluorination substitution on enzyme activity were examined. Remarkably,both TxtE and TxtE-BM3R slightly preferred 5-F-l-tryptophan over1-tryptophan (1.2:1) in the nitration reaction. In addition, althoughthe C4 in 4-F-l-tryptophan is occupied by an F substitution, bothenzymes were able to nitrate this substrate as characterized by HPLC andLC-MS/MS analysis (FIGS. 21A-21C). The overall conversion rate was,however, only about 20% of 1-tryptophan. The F substitution did notinduce any significant change on the UV spectra of substrates andnitrated products (FIG. 22).

Structural Characterization of Fluorinated Nitro-Tryptophan Analogs

Structural characterization of nitrated F-tryptophan products were firstperformed by LC-MS/MS (FIGS. 21A-21C). Nitrated 5-F-l-tryptophan wasfragmented in the same pattern as that of 4-nitro-l-tryptophan in MS2spectra (FIG. 21A-21B). The C₅-F substitution increased the m/z valuesof all corresponding ions by 18 Da and affected the distribution ofdifferent fragments. The most abundant ion in the MS2 spectrum of4-nitro-l-tryptophan had the m/z values of 159.0. It was switched to174.2 in the MS2 spectrum of nitrated 5-F-l-tryptophan, corresponding tothe non-fluorinated ion of 156.2. The most abundant ion in the MS2spectrum of nitro-4-F-1-tryptophan had an m/z value of 209.0 (FIG. 21C).Importantly, its overall fragmentation pattern was notably differentwith that of nitrated 5-F-l-tryptophan. Putative chemical structures ofred-labeled ions in these MS2 spectra are shown in FIG. 23. This resultsuggested that 5-F-1-tryptophan may be nitrated at the same site, theC4, as 1-tryptophan but the nitration site at 4-F-1-tryptophan isdifferent.

To further elucidate the nitro position in nitrated products, largescale enzymatic reactions were performed. About 90% of 5-F-l-tryptophanwas nitrated to produce 2 mg of the nitro product as a yellow powderafter purification by semi-preparative HPLC. In contrast, less than 0.2mg of putative nitro-4-F-l-tryptophan as a light beige solid wasisolated. Both products carried a single nitro group as revealed in HRMSanalysis (FIGS. 24A-24B). Isolated products were further structurallycharacterized by ¹H and ¹³C and 2D NMR analysis (FIGS. 25A, 25B, 26A,26B, 27A, 27B, 28A, and 28B) (Table 2). Examining the NMR data suggestedthat the C4 and the C7 of 5-F-l-tryptophan and 4-F-l-tryptophan,respectively, were nitrated in TxtE-BM3R reactions. From the ¹H NMRspectrum of the nitro 5-F-l-tryptophan product (FIGS. 24A-24B), thelarge coupling constant (J=10.2 Hz) of the triplet-like peak at δ 6.95ppm (C6) suggested a single vicinal coupling with the fluorine atom.Furthermore, a neighboring doublet peak at δ 7.52 ppm (C7) with acoupling constant J=8.8 Hz defined an ortho substitution pattern of thetwo aromatic protons. The aforementioned multiplicity and couplingconstants therefore determined the C4 nitro substitution in the5-F-l-tryptophan substrate (FIG. 14B), which was further confirmed byHSQC and HMBC analysis (FIGS. 27A, 27B, 28A, and 28B). Although <0.2 mgof nitro-4-F-l-tryptophan were isolated, interpretation of the nitroposition in this product was significantly eased using a 1.5 mm HighTemperature Superconductor Probe. From its ¹H NMR spectrum, atriplet-like peak at δ 7.80 ppm (C5) displayed a doublet of doubletsplit with two approximately equal coupling constant of 8.1 Hz,suggesting a vicinal coupling with the fluorine atom. An orthosubstitution pattern of the two aromatic protons was further defined bya large coupling constant (J=9.1 Hz) of the neighboring doublet peak atδ 7.52 ppm (C6). Together, the nitro site was determined to be the C7 of4-F-l-tryptophan (FIG. 14C), which was further confirmed by HSQC andHMBC analysis (FIGS. 27A, 27B, 28A, and 28B). These results thereforerevealed TxtE as a versatile nitrating biocatalyst with remarkablesubstrate promiscuity and substrate-tuned regio-selectivity.

TABLE 3 Determination of total turnover number and coupling efficiencyof TxtE and three fusion enzymes. All reactions were independentlyrepeated at least three times. P450s TTN Coupling efficiency (%) TxtE385 ± 17  2.4 ± 0.3 TxtE-BM3R 321 ± 12  1.9 ± 0.2 TxtE-RhFRed   5 ± 0.3 0.1 ± 0.02 TxtE-RhFRed*   7 ± 0.6  0.3 ± 0.09

The following nitro-tryptophan analogs can be synthesized using any ofthe methods delineated herein, including the processes presented inExamples 1-7.

Example 8: Preparation of(S)-2-amino-3-(5-methyl-4-nitro-1H-indol-3-yl)propanoic acid (8)

Example 8 can be prepared from 5-methylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 9: Preparation of(S)-2-amino-3-(6-methyl-4-nitro-1H-indol-3-yl)propanoic acid (9)

Example 9 can be prepared from 6-methylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 10: Preparation of(S)-2-amino-3-(7-methyl-4-nitro-1H-indol-3-yl)propanoic acid (10)

Example 10 can be prepared from 7-methylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 11: Preparation of(S)-2-amino-3-(4-methyl-7-nitro-1H-indol-3-yl)propanoic acid (11)

Example 11 can be prepared from 4-methylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 12: Preparation of(S)-2-amino-3-(6-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (12)

Example 12 can be prepared from 6-fluoroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 13: Preparation of(S)-2-amino-3-(7-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (13)

Example 13 can be prepared from 7-fluoroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 14: Preparation of(S)-2-amino-3-(5-chloro-4-nitro-1H-indol-3-yl)propanoic acid (14)

Example 14 can be prepared from 5-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 15: Preparation of(S)-2-amino-3-(6-chloro-4-nitro-1H-indol-3-yl)propanoic acid (15)

Example 15 can be prepared from 6-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 16: Preparation of(S)-2-amino-3-(7-chloro-4-nitro-1H-indol-3-yl)propanoic acid (16)

Example 16 can be prepared from 7-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 17: Preparation of(S)-2-amino-3-(4-chloro-7-nitro-1H-indol-3-yl)propanoic acid (17)

Example 17 can be prepared from 4-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 18: Preparation of(S)-2-amino-3-(5-bromo-4-nitro-1H-indol-3-yl)propanoic acid (18)

Example 18 can be prepared from 5-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 19: Preparation of(S)-2-amino-3-(6-bromo-4-nitro-1H-indol-3-yl)propanoic acid (19)

Example 19 can be prepared from 6-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 20: Preparation of(S)-2-amino-3-(7-bromo-4-nitro-1H-indol-3-yl)propanoic acid (20)

Example 20 can be prepared from 7-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 21: Preparation of(S)-2-amino-3-(4-bromo-7-nitro-1H-indol-3-yl)propanoic acid (21)

Example 21 can be prepared from 4-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 22: Preparation of(S)-2-amino-3-(5-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (22)

Example 22 can be prepared from 5-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 23: Preparation of(S)-2-amino-3-(6-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (23)

Example 23 can be prepared from 6-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 24: Preparation of(S)-2-amino-3-(7-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (24)

Example 24 can be prepared from 7-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 25: Preparation of(S)-2-amino-3-(4-methoxy-7-nitro-1H-indol-3-yl)propanoic acid (25)

Example 25 can be prepared from 4-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 26: Preparation of(S)-2-amino-3-(5-amino-4-nitro-1H-indol-3-yl)propanoic acid (26)

Example 26 can be prepared from 5-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 27: Preparation of(S)-2-amino-3-(6-amino-4-nitro-1H-indol-3-yl)propanoic acid (27)

Example 27 can be prepared from 6-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 28: Preparation of(S)-2-amino-3-(7-amino-4-nitro-1H-indol-3-yl)propanoic acid (28)

Example 28 can be prepared from 7-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 29: Preparation of(S)-2-amino-3-(4-amino-7-nitro-1H-indol-3-yl)propanoic acid (29)

Example 29 can be prepared from 4-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 30: Preparation of(S)-2-amino-3-(5-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (30)

Example 30 was prepared from(S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid as shown above andin a similar manner as described in Examples 1-7.

Example 31: Preparation of(S)-2-amino-3-(6-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (31)

Example 31 can be prepared from 6-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 32: Preparation of(S)-2-amino-3-(7-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (32)

Example 32 can be prepared from 7-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 33: Preparation of(S)-2-amino-3-(4-hydroxy-7-nitro-1H-indol-3-yl)propanoic acid (33)

Example 33 can be prepared from 4-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 34: Preparation of(S)-2-amino-3-(4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid (34)

Example 34 can be prepared from 5-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 35: Preparation of(S)-2-amino-3-(4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid (35)

Example 35 can be prepared from 6-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 36: Preparation of(S)-2-amino-3-(4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid (36)

Example 36 can be prepared from 7-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 37: Preparation of(S)-2-amino-3-(7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid (37)

Example 37 can be prepared from 4-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 38: Preparation of(S)-2-amino-3-(5-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (38)

Example 38 can be prepared from 5-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 39: Preparation of(S)-2-amino-3-(6-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (39)

Example 39 can be prepared from 6-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 40: Preparation of(S)-2-amino-3-(7-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (40)

Example 40 can be prepared from 7-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 41: Preparation of(S)-2-amino-3-(4-cyclopropyl-7-nitro-1H-indol-3-yl)propanoic acid (41)

Example 41 can be prepared from 4-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 42: Preparation of(S)-2-amino-3-(4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid (42)

Example 42 can be prepared from 5-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 43: Preparation of(S)-2-amino-3-(4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid (43)

Example 43 can be prepared from 6-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 44: Preparation of(S)-2-amino-3-(4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid (44)

Example 44 can be prepared from 7-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 45: Preparation of(S)-2-amino-3-(7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid (45)

Example 45 can be prepared from 4-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 46: Preparation of(S)-2-amino-3-(5-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (46)

Example 46 can be prepared from 5-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 47: Preparation of(S)-2-amino-3-(6-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (47)

Example 47 can be prepared from 6-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 48: Preparation of(S)-2-amino-3-(7-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (48)

Example 48 can be prepared from 7-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 49: Preparation of(S)-2-amino-3-(4-ethynyl-7-nitro-1H-indol-3-yl)propanoic acid (49)

Example 49 can be prepared from 4-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 50: Preparation of(S)-2-amino-3-(5-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (50)

Example 50 can be prepared from 5-morpholinoindole as shown above and ina similar manner as described in Examples 1-7.

Example 51: Preparation of(S)-2-amino-3-(6-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (51)

Example 51 can be prepared from 6-morpholinoindole as shown above and ina similar manner as described in Examples 1-7.

Example 52: Preparation of(S)-2-amino-3-(7-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (52)

Example 52 can be prepared from 7-morpholinoindole as shown above and ina similar manner as described in Examples 1-7.

Example 53: Preparation of(S)-2-amino-3-(4-morpholino-7-nitro-1H-indol-3-yl)propanoic acid (53)

Example 53 can be prepared from 4-morpholinoindole as shown above and ina similar manner as described in Examples 1-7.

Example 54: Preparation of(S)-2-amino-3-(5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid (54)

Example 54 can be prepared from 5-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 55: Preparation of(S)-2-amino-3-(6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid (55)

Example 55 can be prepared from 6-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 56: Preparation of(S)-2-amino-3-(7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid (56)

Example 56 can be prepared from 7-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 57: Preparation of(S)-2-amino-3-(4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic acid (57)

Example 57 can be prepared from 4-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 58: Preparation of(S)-2-amino-3-(4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid(58)

Example 58 can be prepared from 5-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 59: Preparation of(S)-2-amino-3-(4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid(59)

Example 59 can be prepared from 6-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 60: Preparation of(S)-2-amino-3-(4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid(60)

Example 60 can be prepared from 7-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 61: Preparation of(S)-2-amino-3-(7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid(61)

Example 61 can be prepared from 4-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 62: Preparation of2-amino-3-(5-methyl-4-nitro-1H-indol-3-yl)propanoic acid (62)

Example 62 was prepared from 2-amino-3-(5-methyl-1H-indol-3-yl)propanoicacid as shown above and in a similar manner as described in Examples1-7.

Example 63: Preparation of2-amino-3-(6-methyl-4-nitro-1H-indol-3-yl)propanoic acid (63)

Example 63 can be prepared from 6-methylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 64: Preparation of2-amino-3-(7-methyl-4-nitro-1H-indol-3-yl)propanoic acid (64)

Example 64 was prepared from 2-amino-3-(7-methyl-1H-indol-3-yl)propanoicacid as shown above and in a similar manner as described in Examples1-7.

Example 65: Preparation of2-amino-3-(4-methyl-7-nitro-1H-indol-3-yl)propanoic acid (65)

Example 65 was prepared from 2-amino-3-(4-methyl-1H-indol-3-yl)propanoicacid as shown above and in a similar manner as described in Examples1-7.

Example 66: Preparation of2-amino-3-(6-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (66)

Example 66 was prepared from 2-amino-3-(6-fluoro-1H-indol-3-yl)propanoicacid as shown above and in a similar manner as described in Examples1-7.

Example 67: Preparation of2-amino-3-(7-fluoro-4-nitro-1H-indol-3-yl)propanoic acid (67)

Example 67 can be prepared from 7-fluoroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 68: Preparation of2-amino-3-(4-fluoro-7-nitro-1H-indol-3-yl)propanoic acid (68)

Example 68 was prepared from 2-amino-3-(4-fluoro-1H-indol-3-yl)propanoicacid as shown above and in a similar manner as described in Examples1-7.

Example 69: Preparation of2-amino-3-(5-chloro-4-nitro-1H-indol-3-yl)propanoic acid (69)

Example 69 can be prepared from 5-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 70: Preparation of2-amino-3-(6-chloro-4-nitro-1H-indol-3-yl)propanoic acid (70)

Example 70 can be prepared from 6-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 71: Preparation of2-amino-3-(7-chloro-4-nitro-1H-indol-3-yl)propanoic acid (71)

Example 71 can be prepared from 7-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 72: Preparation of2-amino-3-(4-chloro-7-nitro-1H-indol-3-yl)propanoic acid (72)

Example 72 can be prepared from 4-chloroindole as shown above and in asimilar manner as described in Examples 1-7.

Example 73: Preparation of2-amino-3-(5-bromo-4-nitro-1H-indol-3-yl)propanoic acid (73)

Example 73 can be prepared from 5-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 74: Preparation of2-amino-3-(6-bromo-4-nitro-1H-indol-3-yl)propanoic acid (74)

Example 74 can be prepared from 6-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 75: Preparation of2-amino-3-(7-bromo-4-nitro-H-indol-3-yl)propanoic acid (75)

Example 75 can be prepared from 7-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 76: Preparation of2-amino-3-(4-bromo-7-nitro-1H-indol-3-yl)propanoic acid (76)

Example 76 can be prepared from 4-bromoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 77: Preparation of2-amino-3-(5-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (77)

Example 77 can be prepared from 5-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 78: Preparation of2-amino-3-(6-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (78)

Example 78 can be prepared from 6-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 79: Preparation of2-amino-3-(7-methoxy-4-nitro-1H-indol-3-yl)propanoic acid (79)

Example 79 can be prepared from 7-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 80: Preparation of2-amino-3-(4-methoxy-7-nitro-1H-indol-3-yl)propanoic acid (80)

Example 80 can be prepared from 4-methoxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 81: Preparation of2-amino-3-(5-amino-4-nitro-1H-indol-3-yl)propanoic acid (81)

Example 81 can be prepared from 5-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 82: Preparation of2-amino-3-(6-amino-4-nitro-1H-indol-3-yl)propanoic acid (82)

Example 82 can be prepared from 6-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 83: Preparation of2-amino-3-(7-amino-4-nitro-1H-indol-3-yl)propanoic acid (83)

Example 83 can be prepared from 7-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 84: Preparation of2-amino-3-(4-amino-7-nitro-1H-indol-3-yl)propanoic acid (84)

Example 84 can be prepared from 4-aminoindole as shown above and in asimilar manner as described in Examples 1-7.

Example 85: Preparation of2-amino-3-(5-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (85)

Example 85 can be prepared from 5-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 86: Preparation of2-amino-3-(6-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (86)

Example 86 can be prepared from 6-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 87: Preparation of2-amino-3-(7-hydroxy-4-nitro-1H-indol-3-yl)propanoic acid (87)

Example 87 can be prepared from 7-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 88: Preparation of2-amino-3-(4-hydroxy-7-nitro-1H-indol-3-yl)propanoic acid (88)

Example 88 can be prepared from 4-hydroxyindole as shown above and in asimilar manner as described in Examples 1-7.

Example 89: Preparation of2-amino-3-(4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid (89)

Example 89 can be prepared from 5-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 90: Preparation of2-amino-3-(4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid (90)

Example 90 can be prepared from 6-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 91: Preparation of2-amino-3-(4-nitro-7-phenyl-H-indol-3-yl)propanoic acid (91)

Example 91 can be prepared from 7-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 92: Preparation of2-amino-3-(7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid (92)

Example 92 can be prepared from 4-phenylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 93: Preparation of2-amino-3-(5-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (93)

Example 93 can be prepared from 5-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 94: Preparation of2-amino-3-(6-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (94)

Example 94 can be prepared from 6-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 95: Preparation of2-amino-3-(7-cyclopropyl-4-nitro-1H-indol-3-yl)propanoic acid (95)

Example 95 can be prepared from 7-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 96: Preparation of2-amino-3-(4-cyclopropyl-7-nitro-1H-indol-3-yl)propanoic acid (96)

Example 96 can be prepared from 4-cyclopropylindole as shown above andin a similar manner as described in Examples 1-7.

Example 97: Preparation of2-amino-3-(4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid (97)

Example 97 can be prepared from 5-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 98: Preparation of2-amino-3-(4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid (98)

Example 98 can be prepared from 6-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 99: Preparation of2-amino-3-(4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid (99)

Example 99 can be prepared from 7-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 100: Preparation of2-amino-3-(7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid (100)

Example 100 can be prepared from 4-vinylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 101: Preparation of2-amino-3-(5-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (101)

Example 101 can be prepared from 5-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 102: Preparation of2-amino-3-(6-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (102)

Example 102 can be prepared from 6-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 103: Preparation of2-amino-3-(7-ethynyl-4-nitro-1H-indol-3-yl)propanoic acid (103)

Example 103 can be prepared from 7-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 104: Preparation of2-amino-3-(4-ethynyl-7-nitro-1H-indol-3-yl)propanoic acid (104)

Example 104 can be prepared from 4-ethynylindole as shown above and in asimilar manner as described in Examples 1-7.

Example 105: Preparation of2-amino-3-(5-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (105)

Example 105 can be prepared from 5-morpholinoindole as shown above andin a similar manner as described in Examples 1-7.

Example 106: Preparation of2-amino-3-(6-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (106)

Example 106 can be prepared from 6-morpholinoindole as shown above andin a similar manner as described in Examples 1-7.

Example 107: Preparation of2-amino-3-(7-morpholino-4-nitro-1H-indol-3-yl)propanoic acid (107)

Example 107 can be prepared from 7-morpholinoindole as shown above andin a similar manner as described in Examples 1-7.

Example 108: Preparation of2-amino-3-(4-morpholino-7-nitro-1H-indol-3-yl)propanoic acid (108)

Example 108 can be prepared from 4-morpholinoindole as shown above andin a similar manner as described in Examples 1-7.

Example 109: Preparation of2-amino-3-(5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid (109)

Example 109 can be prepared from 5-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 110: Preparation of2-amino-3-(6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid (110)

Example 110 can be prepared from 6-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 111: Preparation of2-amino-3-(7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid (111)

Example 111 can be prepared from 7-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 112: Preparation of2-amino-3-(4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic acid (112)

Example 112 can be prepared from 4-(methylthio)indole as shown above andin a similar manner as described in Examples 1-7.

Example 113: Preparation of2-amino-3-(4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid (113)

Example 113 can be prepared from 5-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 114: Preparation of2-amino-3-(4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid (114)

Example 114 can be prepared from 6-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 115: Preparation of2-amino-3-(4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid (115)

Example 115 can be prepared from 7-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 116: Preparation of2-amino-3-(7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoic acid (116)

Example 116 can be prepared from 4-(pyridin-4-yl)indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 117: Preparation of2-amino-3-(1,5-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (117)

Example 117 can be prepared from 1,5-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 118: Preparation of2-amino-3-(1,6-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (118)

Example 118 can be prepared from 1,6-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 119: Preparation of 2-amino-3-(1,7-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (119)

Example 119 can be prepared from 1,7-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 120: Preparation of2-amino-3-(1,4-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid (120)

Example 120 can be prepared from 1,4-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 121: Preparation of2-amino-3-(6-fluoro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (121)

Example 121 can be prepared from 6-fluoro-1-methyl-1H-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 122: Preparation of2-amino-3-(7-fluoro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (122)

Example 122 can be prepared from 7-fluoro-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 123: Preparation of2-amino-3-(4-fluoro-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (123)

Example 123 can be prepared from 4-fluoro-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 124: Preparation of2-amino-3-(5-chloro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (124)

Example 124 can be prepared from 5-chloro-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 125: Preparation of2-amino-3-(6-chloro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (125)

Example 125 can be prepared from 6-chloro-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 126: Preparation of2-amino-3-(7-chloro-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (126)

Example 126 can be prepared from 7-chloro-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 127: Preparation of2-amino-3-(4-chloro-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (127)

Example 127 can be prepared from 4-chloro-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 128: Preparation of2-amino-3-(5-bromo-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (128)

Example 128 can be prepared from 5-bromo-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 129: Preparation of2-amino-3-(6-bromo-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (129)

Example 129 can be prepared from 6-bromo-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 130: Preparation of2-amino-3-(7-bromo-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (130)

Example 130 can be prepared from 7-bromo-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 131: Preparation of2-amino-3-(4-bromo-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (131)

Example 131 can be prepared from 4-bromo-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 132: Preparation of2-amino-3-(5-methoxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (132)

Example 132 can be prepared from 5-methoxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 133: Preparation of2-amino-3-(6-methoxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (133)

Example 133 can be prepared from 6-methoxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 134: Preparation of2-amino-3-(7-methoxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (134)

Example 134 can be prepared from 7-methoxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 135: Preparation of2-amino-3-(4-methoxy-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (135)

Example 135 can be prepared from 4-methoxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 136: Preparation of2-amino-3-(5-amino-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (136)

Example 136 can be prepared from 5-amino-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 137: Preparation of2-amino-3-(6-amino-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (137)

Example 137 can be prepared from 6-amino-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 138: Preparation of2-amino-3-(7-amino-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (138)

Example 138 can be prepared from 7-amino-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 139: Preparation of2-amino-3-(4-amino-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (139)

Example 139 can be prepared from 4-amino-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 140: Preparation of2-amino-3-(5-hydroxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (140)

Example 140 can be prepared from 5-hydroxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 141: Preparation of2-amino-3-(6-hydroxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (141)

Example 141 can be prepared from 6-hydroxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 142: Preparation of2-amino-3-(7-hydroxy-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (142)

Example 142 can be prepared from 7-hydroxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 143: Preparation of2-amino-3-(4-hydroxy-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (143)

Example 143 can be prepared from 4-hydroxy-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 144: Preparation of2-amino-3-(1-methyl-4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid (144)

Example 144 can be prepared from 5-phenyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 145: Preparation of2-amino-3-(1-methyl-4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid (145)

Example 145 can be prepared from 6-phenyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 146: Preparation of2-amino-3-(1-methyl-4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid (146)

Example 146 can be prepared from 7-phenyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 147: Preparation of2-amino-3-(1-methyl-7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid (147)

Example 147 can be prepared from 4-phenyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 148: Preparation of2-amino-3-(5-cyclopropyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid(148)

Example 148 can be prepared from 5-cyclopropyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 149: Preparation of2-amino-3-(6-cyclopropyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid(149)

Example 149 can be prepared from 6-cyclopropyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 150: Preparation of2-amino-3-(7-cyclopropyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid(150)

Example 150 can be prepared from 7-cyclopropyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 151: Preparation of2-amino-3-(4-cyclopropyl-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid(151)

Example 151 can be prepared from 4-cyclopropyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 152: Preparation of2-amino-3-(1-methyl-4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid (152)

Example 152 can be prepared from 5-vinyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 153: Preparation of2-amino-3-(1-methyl-4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid (153)

Example 153 can be prepared from 6-vinyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 154: Preparation of2-amino-3-(1-methyl-4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid (154)

Example 154 can be prepared from 7-vinyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 155: Preparation of2-amino-3-(1-methyl-7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid (155)

Example 155 can be prepared from 4-vinyl-1-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 156: Preparation of2-amino-3-(5-ethynyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (156)

Example 156 can be prepared from 5-ethynyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 157: Preparation of2-amino-3-(6-ethynyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (157)

Example 157 can be prepared from 6-ethynyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 158: Preparation of2-amino-3-(7-ethynyl-1-methyl-4-nitro-1H-indol-3-yl)propanoic acid (158)

Example 158 can be prepared from 7-ethynyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 159: Preparation of2-amino-3-(4-ethynyl-1-methyl-7-nitro-1H-indol-3-yl)propanoic acid (159)

Example 159 can be prepared from 4-ethynyl-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 160: Preparation of2-amino-3-(1-methyl-5-morpholino-4-nitro-1H-indol-3-yl)propanoic acid(160)

Example 160 can be prepared from 5-morpholino-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 161: Preparation of2-amino-3-(1-methyl-6-morpholino-4-nitro-1H-indol-3-yl)propanoic acid(161)

Example 161 can be prepared from 6-morpholino-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 162: Preparation of2-amino-3-(1-methyl-7-morpholino-4-nitro-1H-indol-3-yl)propanoic acid(162)

Example 162 can be prepared from 7-morpholino-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 163: Preparation of2-amino-3-(1-methyl-4-morpholino-7-nitro-1H-indol-3-yl)propanoic acid(163)

Example 163 can be prepared from 4-morpholino-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 164: Preparation of2-amino-3-(1-methyl-5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid(164)

Example 164 can be prepared from 5-(methylthio)-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 165: Preparation of2-amino-3-(1-methyl-6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid(165)

Example 165 can be prepared from 6-(methylthio)-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 166: Preparation of2-amino-3-(1-methyl-7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid(166)

Example 166 can be prepared from 7-(methylthio)-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 167: Preparation of2-amino-3-(1-methyl-4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic acid(167)

Example 167 can be prepared from 4-(methylthio)-1-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 168: Preparation of2-amino-3-(1-methyl-4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (168)

Example 168 can be prepared from 5-(pyridin-4-yl)-1-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 169: Preparation of2-amino-3-(1-methyl-4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (169)

Example 169 can be prepared from 6-(pyridin-4-yl)-1-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 170: Preparation of2-amino-3-(1-methyl-4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (170)

Example 170 can be prepared from 7-(pyridin-4-yl)-1-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 171: Preparation of2-amino-3-(1-methyl-7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (171)

Example 171 can be prepared from 4-(pyridin-4-yl)-1-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 172: Preparation of2-amino-3-(2,5-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (172)

Example 172 can be prepared from 2,5-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 173: Preparation of2-amino-3-(2,6-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (173)

Example 173 can be prepared from 2,6-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 174: Preparation of 2-amino-3-(2,7-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid (174)

Example 174 can be prepared from 2,7-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 175: Preparation of2-amino-3-(2,4-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid (175)

Example 175 can be prepared from 2,4-dimethyl-1H-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 176: Preparation of2-amino-3-(6-fluoro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (176)

Example 176 can be prepared from 6-fluoro-2-methyl-1H-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 177: Preparation of2-amino-3-(7-fluoro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (177)

Example 177 can be prepared from 7-fluoro-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 178: Preparation of2-amino-3-(4-fluoro-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (178)

Example 178 can be prepared from 4-fluoro-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 179: Preparation of2-amino-3-(5-chloro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (179)

Example 179 can be prepared from 5-chloro-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 180: Preparation of2-amino-3-(6-chloro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (180)

Example 180 can be prepared from 6-chloro-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 181: Preparation of2-amino-3-(7-chloro-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (181)

Example 181 can be prepared from 7-chloro-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 182: Preparation of2-amino-3-(4-chloro-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (182)

Example 182 can be prepared from 4-chloro-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 183: Preparation of2-amino-3-(5-bromo-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (183)

Example 183 can be prepared from 5-bromo-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 184: Preparation of2-amino-3-(6-bromo-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (184)

Example 184 can be prepared from 6-bromo-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 185: Preparation of2-amino-3-(7-bromo-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (185)

Example 185 can be prepared from 7-bromo-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 186: Preparation of2-amino-3-(4-bromo-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (186)

Example 186 can be prepared from 4-bromo-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 187: Preparation of2-amino-3-(5-methoxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (187)

Example 187 can be prepared from 5-methoxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 188: Preparation of2-amino-3-(6-methoxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (188)

Example 188 can be prepared from 6-methoxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 189: Preparation of2-amino-3-(7-methoxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (189)

Example 189 can be prepared from 7-methoxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 190: Preparation of2-amino-3-(4-methoxy-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (190)

Example 190 can be prepared from 4-methoxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 191: Preparation of2-amino-3-(5-amino-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (191)

Example 191 can be prepared from 5-amino-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 192: Preparation of2-amino-3-(6-amino-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (192)

Example 192 can be prepared from 6-amino-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 193: Preparation of2-amino-3-(7-amino-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (193)

Example 193 can be prepared from 7-amino-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 194: Preparation of2-amino-3-(4-amino-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (194)

Example 194 can be prepared from 4-amino-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 195: Preparation of2-amino-3-(5-hydroxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (195)

Example 195 can be prepared from 5-hydroxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 196: Preparation of2-amino-3-(6-hydroxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (196)

Example 196 can be prepared from 6-hydroxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 197: Preparation of2-amino-3-(7-hydroxy-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (197)

Example 197 can be prepared from 7-hydroxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 198: Preparation of2-amino-3-(4-hydroxy-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (198)

Example 198 can be prepared from 4-hydroxy-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 199: Preparation of2-amino-3-(2-methyl-4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid (199)

Example 199 can be prepared from 5-phenyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 200: Preparation of2-amino-3-(2-methyl-4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid (200)

Example 200 can be prepared from 6-phenyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 201: Preparation of2-amino-3-(2-methyl-4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid (201)

Example 201 can be prepared from 7-phenyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 202: Preparation of2-amino-3-(2-methyl-7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid (202)

Example 202 can be prepared from 4-phenyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 203: Preparation of2-amino-3-(5-cyclopropyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid(203)

Example 203 can be prepared from 5-cyclopropyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 204: Preparation of2-amino-3-(6-cyclopropyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid(204)

Example 204 can be prepared from 6-cyclopropyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 205: Preparation of2-amino-3-(7-cyclopropyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid(205)

Example 205 can be prepared from 7-cyclopropyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 206: Preparation of2-amino-3-(4-cyclopropyl-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid(206)

Example 206 can be prepared from 4-cyclopropyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 207: Preparation of2-amino-3-(2-methyl-4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid (207)

Example 207 can be prepared from 5-vinyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 208: Preparation of2-amino-3-(2-methyl-4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid (208)

Example 208 can be prepared from 6-vinyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 209: Preparation of2-amino-3-(2-methyl-4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid (209)

Example 209 can be prepared from 7-vinyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 210: Preparation of2-amino-3-(2-methyl-7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid (210)

Example 210 can be prepared from 4-vinyl-2-methyl-indole as shown aboveand in a similar manner as described in Examples 1-7.

Example 211: Preparation of2-amino-3-(5-ethynyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (211)

Example 211 can be prepared from 5-ethynyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 212: Preparation of2-amino-3-(6-ethynyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (212)

Example 212 can be prepared from 6-ethynyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 213: Preparation of2-amino-3-(7-ethynyl-2-methyl-4-nitro-1H-indol-3-yl)propanoic acid (213)

Example 213 can be prepared from 7-ethynyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 214: Preparation of2-amino-3-(4-ethynyl-2-methyl-7-nitro-1H-indol-3-yl)propanoic acid (214)

Example 214 can be prepared from 4-ethynyl-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 215: Preparation of2-amino-3-(2-methyl-5-morpholino-4-nitro-1H-indol-3-yl)propanoic acid(215)

Example 215 can be prepared from 5-morpholino-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 216: Preparation of2-amino-3-(2-methyl-6-morpholino-4-nitro-1H-indol-3-yl)propanoic acid(216)

Example 216 can be prepared from 6-morpholino-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 217: Preparation of2-amino-3-(2-methyl-7-morpholino-4-nitro-1H-indol-3-yl)propanoic acid(217)

Example 217 can be prepared from 7-morpholino-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 218: Preparation of2-amino-3-(2-methyl-4-morpholino-7-nitro-1H-indol-3-yl)propanoic acid(218)

Example 218 can be prepared from 4-morpholino-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 219: Preparation of2-amino-3-(2-methyl-5-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid(219)

Example 219 can be prepared from 5-(methylthio)-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 220: Preparation of2-amino-3-(2-methyl-6-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid(220)

Example 220 can be prepared from 6-(methylthio)-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 221: Preparation of2-amino-3-(2-methyl-7-(methylthio)-4-nitro-1H-indol-3-yl)propanoic acid(221)

Example 221 can be prepared from 7-(methylthio)-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 222: Preparation of2-amino-3-(2-methyl-4-(methylthio)-7-nitro-1H-indol-3-yl)propanoic acid(222)

Example 222 can be prepared from 4-(methylthio)-2-methyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 223: Preparation of2-amino-3-(2-methyl-4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (223)

Example 223 can be prepared from 5-(pyridin-4-yl)-2-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 224: Preparation of2-amino-3-(2-methyl-4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (224)

Example 224 can be prepared from 6-(pyridin-4-yl)-2-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 225: Preparation of2-amino-3-(2-methyl-4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (225)

Example 225 can be prepared from 7-(pyridin-4-yl)-2-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 226: Preparation of2-amino-3-(2-methyl-7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (226)

Example 226 can be prepared from 4-(pyridin-4-yl)-2-methyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 227: Preparation of2-amino-3-(1,2,5-trimethyl-4-nitro-1H-indol-3-yl)propanoic acid (227)

Example 227 can be prepared from 1,2,5-trimethyl-1H-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 228: Preparation of2-amino-3-(1,2,6-trimethyl-4-nitro-1H-indol-3-yl)propanoic acid (228)

Example 228 can be prepared from 1,2,6-trimethyl-1H-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 229: Preparation of 2-amino-3-(1,2,7-trimethyl-4-nitro-1H-indol-3-yl)propanoic acid (229)

Example 229 can be prepared from 1,2,7-trimethyl-1H-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 230: Preparation of2-amino-3-(1,2,4-trimethyl-7-nitro-1H-indol-3-yl)propanoic acid (230)

Example 230 can be prepared from 1,2,4-trimethyl-1H-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 231: Preparation of2-amino-3-(6-fluoro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(231)

Example 231 can be prepared from 6-fluoro-1,2-dimethyl-1H-indole asshown above and in a similar manner as described in Examples 1-7.

Example 232: Preparation of2-amino-3-(7-fluoro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(232)

Example 232 can be prepared from 7-fluoro-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 233: Preparation of2-amino-3-(4-fluoro-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(233)

Example 233 can be prepared from 4-fluoro-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 234: Preparation of2-amino-3-(5-chloro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(234)

Example 234 can be prepared from 5-chloro-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 235: Preparation of2-amino-3-(6-chloro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(235)

Example 235 can be prepared from 6-chloro-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 236: Preparation of2-amino-3-(7-chloro-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(236)

Example 236 can be prepared from 7-chloro-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 237: Preparation of2-amino-3-(4-chloro-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(237)

Example 237 can be prepared from 4-chloro-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 238: Preparation of2-amino-3-(5-bromo-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(238)

Example 238 can be prepared from 5-bromo-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 239: Preparation of2-amino-3-(6-bromo-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(239)

Example 239 can be prepared from 6-bromo-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 240: Preparation of2-amino-3-(7-bromo-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(240)

Example 240 can be prepared from 7-bromo-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 241: Preparation of2-amino-3-(4-bromo-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(241)

Example 241 can be prepared from 4-bromo-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 242: Preparation of2-amino-3-(5-methoxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(242)

Example 242 can be prepared from 5-methoxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 243: Preparation of2-amino-3-(6-methoxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(243)

Example 243 can be prepared from 6-methoxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 244: Preparation of2-amino-3-(7-methoxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(244)

Example 244 can be prepared from 7-methoxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 245: Preparation of2-amino-3-(4-methoxy-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(245)

Example 245 can be prepared from 4-methoxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 246: Preparation of2-amino-3-(5-amino-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(246)

Example 246 can be prepared from 5-amino-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 247: Preparation of2-amino-3-(6-amino-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(247)

Example 247 can be prepared from 6-amino-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 248: Preparation of2-amino-3-(7-amino-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(248)

Example 248 can be prepared from 7-amino-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 249: Preparation of2-amino-3-(4-amino-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(249)

Example 249 can be prepared from 4-amino-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 250: Preparation of2-amino-3-(5-hydroxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(250)

Example 250 can be prepared from 5-hydroxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 251: Preparation of2-amino-3-(6-hydroxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(251)

Example 251 can be prepared from 6-hydroxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 252: Preparation of2-amino-3-(7-hydroxy-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(252)

Example 252 can be prepared from 7-hydroxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 253: Preparation of2-amino-3-(4-hydroxy-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(253)

Example 253 can be prepared from 4-hydroxy-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 254: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-5-phenyl-1H-indol-3-yl)propanoic acid(254)

Example 254 can be prepared from 5-phenyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 255: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-6-phenyl-1H-indol-3-yl)propanoic acid(255)

Example 255 can be prepared from 6-phenyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 256: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-7-phenyl-1H-indol-3-yl)propanoic acid(256)

Example 256 can be prepared from 7-phenyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 257: Preparation of2-amino-3-(1,2-dimethyl-7-nitro-4-phenyl-1H-indol-3-yl)propanoic acid(257)

Example 257 can be prepared from 4-phenyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 258: Preparation of2-amino-3-(5-cyclopropyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoicacid (258)

Example 258 can be prepared from 5-cyclopropyl-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 259: Preparation of2-amino-3-(6-cyclopropyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoicacid (259)

Example 259 can be prepared from 6-cyclopropyl-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 260: Preparation of2-amino-3-(7-cyclopropyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoicacid (260)

Example 260 can be prepared from 7-cyclopropyl-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 261: Preparation of2-amino-3-(4-cyclopropyl-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoicacid (261)

Example 261 can be prepared from 4-cyclopropyl-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 262: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-5-vinyl-1H-indol-3-yl)propanoic acid(262)

Example 262 can be prepared from 5-vinyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 263: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-6-vinyl-1H-indol-3-yl)propanoic acid(263)

Example 263 can be prepared from 6-vinyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 264: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-7-vinyl-1H-indol-3-yl)propanoic acid(264)

Example 264 can be prepared from 7-vinyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 265: Preparation of2-amino-3-(1,2-dimethyl-7-nitro-4-vinyl-1H-indol-3-yl)propanoic acid(265)

Example 265 can be prepared from 4-vinyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 266: Preparation of2-amino-3-(5-ethynyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(266)

Example 266 can be prepared from 5-ethynyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 267: Preparation of2-amino-3-(6-ethynyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(267)

Example 267 can be prepared from 6-ethynyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 268: Preparation of2-amino-3-(7-ethynyl-1,2-dimethyl-4-nitro-1H-indol-3-yl)propanoic acid(268)

Example 268 can be prepared from 7-ethynyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 269: Preparation of2-amino-3-(4-ethynyl-1,2-dimethyl-7-nitro-1H-indol-3-yl)propanoic acid(269)

Example 269 can be prepared from 4-ethynyl-1,2-dimethyl-indole as shownabove and in a similar manner as described in Examples 1-7.

Example 270: Preparation of2-amino-3-(1,2-dimethyl-5-morpholino-4-nitro-1H-indol-3-yl)propanoicacid (270)

Example 270 can be prepared from 5-morpholino-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 271: Preparation of2-amino-3-(1,2-dimethyl-6-morpholino-4-nitro-1H-indol-3-yl)propanoicacid (271)

Example 271 can be prepared from 6-morpholino-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 272: Preparation of2-amino-3-(1,2-dimethyl-7-morpholino-4-nitro-1H-indol-3-yl)propanoicacid (272)

Example 272 can be prepared from 7-morpholino-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 273: Preparation of2-amino-3-(1,2-dimethyl-4-morpholino-7-nitro-1H-indol-3-yl)propanoicacid (273)

Example 273 can be prepared from 4-morpholino-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 274: Preparation of2-amino-3-(1,2-dimethyl-5-(methylthio)-4-nitro-1H-indol-3-yl)propanoicacid (274)

Example 274 can be prepared from 5-(methylthio)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 275: Preparation of2-amino-3-(1,2-dimethyl-6-(methylthio)-4-nitro-1H-indol-3-yl)propanoicacid (275)

Example 275 can be prepared from 6-(methylthio)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 276: Preparation of2-amino-3-(1,2-dimethyl-7-(methylthio)-4-nitro-1H-indol-3-yl)propanoicacid (276)

Example 276 can be prepared from 7-(methylthio)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 277: Preparation of2-amino-3-(1,2-dimethyl-4-(methylthio)-7-nitro-1H-indol-3-yl)propanoicacid (277)

Example 277 can be prepared from 4-(methylthio)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 278: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-5-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (278)

Example 278 can be prepared from 5-(pyridin-4-yl)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 279: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-6-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (279)

Example 279 can be prepared from 6-(pyridin-4-yl)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 280: Preparation of2-amino-3-(1,2-dimethyl-4-nitro-7-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (280)

Example 280 can be prepared from 7-(pyridin-4-yl)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

Example 281: Preparation of2-amino-3-(1,2-dimethyl-7-nitro-4-(pyridin-4-yl)-1H-indol-3-yl)propanoicacid (281)

Example 281 can be prepared from 4-(pyridin-4-yl)-1,2-dimethyl-indole asshown above and in a similar manner as described in Examples 1-7.

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1-69. (canceled)
 70. A cell comprising a fusion protein, wherein thefusion protein comprises: (i) a cytochrome P450 enzyme which catalyzestransfer of a nitro functional group to provide a compound representedby Formula V:

wherein: X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), wherein R^(Ala) is independently hydrogen,substituted or unsubstituted acyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a nitrogen protecting groupwhen attached to a nitrogen atom, an oxygen protecting group whenattached to an oxygen atom, or a sulfur protecting group when attachedto a sulfur atom, or two instances of R^(Ala) are joined to form asubstituted or unsubstituted, heterocyclic ring, or substituted orunsubstituted, heteroaryl ring; each of X² and X³ is, independently,hydrogen, halogen, substituted or unsubstituted C₁₋₆ alkyl, substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), wherein R^(Ala) is independently hydrogen,substituted or unsubstituted acyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a nitrogen protecting groupwhen attached to a nitrogen atom, an oxygen protecting group whenattached to an oxygen atom, or a sulfur protecting group when attachedto a sulfur atom, or two instances of R^(Ala) are joined to form asubstituted or unsubstituted, heterocyclic ring, or substituted orunsubstituted, heteroaryl ring; R₁ is H or optionally substituted alkyl;R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl; and Y is NO₂; or a pharmaceuticallyacceptable salt thereof; (ii) an amino acid linker; and, (iii) acatalytic domain of a reductase enzyme; wherein the linker joins (iii)to a terminus of (i).
 71. The cell of claim 70, wherein the P450 enzymeoccurs naturally in Streptomyces.
 72. The cell of claim 70, wherein theP450 enzyme is a TxtE enzyme, wherein a TxtE enzyme is defined as: (i)TxtE; (ii) a portion of TxtE which catalyzes transfer of a nitrofunctional group to provide a compound represented by Formula V:

wherein: X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), wherein R^(Ala) is independently hydrogen,substituted or unsubstituted acyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a nitrogen protecting groupwhen attached to a nitrogen atom, an oxygen protecting group whenattached to an oxygen atom, or a sulfur protecting group when attachedto a sulfur atom, or two instances of R^(Ala) are joined to form asubstituted or unsubstituted, heterocyclic ring, or substituted orunsubstituted, heteroaryl ring; each of X² and X³ is, independently,hydrogen, halogen, substituted or unsubstituted C₁₋₆ alkyl, substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), wherein R^(Ala) is independently hydrogen,substituted or unsubstituted acyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a nitrogen protecting groupwhen attached to a nitrogen atom, an oxygen protecting group whenattached to an oxygen atom, or a sulfur protecting group when attachedto a sulfur atom, or two instances of R^(Ala) are joined to form asubstituted or unsubstituted, heterocyclic ring, or substituted orunsubstituted, heteroaryl ring; R₁ is H or optionally substituted alkyl;R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl; and Y is NO₂; or a pharmaceuticallyacceptable salt thereof; or, (iii) an enzyme which is at least 95%homologous to the amino acid sequence of TxtE and which catalyzestransfer of a nitro functional group to provide a compound representedby Formula V:

wherein: X¹ is halogen, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted C₂₋₆ alkenyl, substituted or unsubstitutedC₂₋₆ alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), wherein R^(Ala) is independently hydrogen,substituted or unsubstituted acyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a nitrogen protecting groupwhen attached to a nitrogen atom, an oxygen protecting group whenattached to an oxygen atom, or a sulfur protecting group when attachedto a sulfur atom, or two instances of R^(Ala) are joined to form asubstituted or unsubstituted, heterocyclic ring, or substituted orunsubstituted, heteroaryl ring; each of X² and X³ is, independently,hydrogen, halogen, substituted or unsubstituted C₁₋₆ alkyl, substitutedor unsubstituted C₂₋₆ alkenyl, substituted or unsubstituted C₂₋₆alkynyl, substituted or unsubstituted, monocyclic, 3-to 6-memberedcarbocyclyl, substituted or unsubstituted, monocyclic, 3- to 6-memberedheterocyclyl, substituted or unsubstituted phenyl, substituted orunsubstituted, monocyclic, 5- to 6-membered heteroaryl, —OR^(Ala),—N(R^(Ala))₂, or —SR^(Ala), wherein R^(Ala) is independently hydrogen,substituted or unsubstituted acyl, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a nitrogen protecting groupwhen attached to a nitrogen atom, an oxygen protecting group whenattached to an oxygen atom, or a sulfur protecting group when attachedto a sulfur atom, or two instances of R^(Ala) are joined to form asubstituted or unsubstituted, heterocyclic ring, or substituted orunsubstituted, heteroaryl ring; R₁ is H or optionally substituted alkyl;R₂ is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, oroptionally substituted heteroaryl; and Y is NO₂; or a pharmaceuticallyacceptable salt thereof.
 73. The cell of claim 70, wherein thecytochrome P450 enzyme shares at least 90% amino acid sequencesimilarity with TxtE.
 74. The cell of claim 70, wherein the cell is aprokaryotic cell.
 75. The cell of claim 74, wherein the prokaryotic cellis a bacterial cell.
 76. The cell of claim 75, wherein the bacterialcell is an E. coli cell.
 77. The cell of claim 70, wherein the fusionprotein is encoded by an expression construct.
 78. The cell of claim 77,wherein the expression construct is a plasmid.